Methods for substantially simultaneous evaluation of a sample containing a cellular target and a soluble analyte

ABSTRACT

Methods are provided for monitoring treatment of a subject using heparin therapy or a ligand, such as an CD20 or CD52 antibody, that binds to a cellular marker and to which the subject may develop masking reactions or auto-antibodies that inhibit evaluation of treatment. The methods involve adding to a single container a sample containing cells that express the cellular target with (i) a soluble ligand that binds the cellular target, (ii) a soluble ligand that binds the soluble analyte or a competing soluble analyte; and (iii) a capture medium that binds directly to the soluble analyte, indirectly to the soluble analyte, or to the soluble ligand that binds to the soluble analyte. Complexes formed in the container by interaction of these components are substantially simultaneously analyzed and quantitated without physically separating the complexes. Kits for performing the assays are also provided.

BACKGROUND OF THE INVENTION

The present invention relates generally to assay methods forquantitative and qualitative evaluation of biological samples and moreparticularly to assay methods for biological samples containing bothcellular and soluble targets or analytes.

The ability to detect and/or measure a wide variety of targets,analytes, molecules, chemical compounds and complexes and the like in avariety of biological samples or products has significant use thediagnosis of disease, the treatment of disease, the monitoring of theefficacy of therapy, research in molecular biology, the detection andmonitoring of water purity, product contamination, and other fields. Anumber of different types of assays have evolved depending upon theidentity and state of the target to be analyzed, e.g., a targetimmobilized on or in a substrate (such as a cell) or a soluble target, atarget that is proteinaceous, or a chemical compound, etc. Thus,immunoassays exist that identify proteinaceous targets using antibodies;competition immunoassays identify targets by allowing the target tocompete for binding to a limited amount of an antibody with a knownamount of a labeled antigen. The amount of labeled antigen bound to theantibody is inversely proportional to the amount of antigen in thesample. Immunometric assays employ a labeled antibody and the amount oflabeled antibody associated with the target is measured as directlyproportional to the amount of target available in the sample. Cytometricassays identify targets by size, shape, charge, light diffraction orreflection, or other means.

Generally, assays to detect targets by immobilizing a ligand on a solidsupport and forming a complex between the target and the bound ligandrequire different processing steps than assays employing soluble ligandsto detect. Further, in assays performed on blood samples, phagocytosisof the solid support particles by myeloid cells in the sample canintroduce error into the assays. Most known assays also involve multipleprocessing steps, e.g., lysing, washing, and physically separatingcomponents formed in the sample prior to detecting the appropriateligand or label. Further known assays generally employ a wash step toremove bound analyte from soluble analyte and are performed in a serum,plasma or media matrix devoid of cells.

Additional steps of washing, lysing and separating introduceinaccuracies by reducing the amount of target inadvertently. The need toperform a number of different assays on a single rare or small sample isoften another problem experienced with assays, which leads to depletionof the sample material and an increase in the costs of the assaysthemselves.

Various assays are described in the following references: Lindmo T, etal, J. Immunol. Meth., 1990 126:183-189; Frengen J et al, J. Immunol.Meth., 1995 178:131-140; Frengen J et al, J. Immunol. Meth., 1995178:141-151; U.S. Pat. Nos. 4,572,028; 4,376,110; 5,006,459; 5,168,044;5,426,029; 5,525,461; 5,567,627; 5,756,011; 5,811,525; 5,981,180;6,268,155; and U.S. patent publication 2002/0076833.

Recent approvals of biologic therapies for treatment of cancer haveopened up a new area for therapeutic monitoring. For example, treatmentof B-cell lymphoma with monoclonal antibodies directed against CD20(Rituximab®, Bexxar™) was FDA approved a few years ago. Initial resultshave been promising, however continued monitoring to evaluate remissionversus tumor escape is required. After treatment, B-cells may no longerexpress CD20 due to B-cell depletion, tumor escape, or blocking of theCD20 either by the anti-CD20 treatment antibody (Rituximab, or Bexxar)or by circulating soluble CD20 that binds the detector antibody (Giles,F. J., et al. 2003. Br J Haematol 123:850-857).

For example, levels of circulating Rituximab® occur up to 6 monthspost-treatment and can block detection of CD20+ cells. The presence ofcytoplasmic CD20 will indicate that Rituximab® is effectively blockingB-cell CD20, not that the B-cells have become CD20 negative (tumorescape) (Clarke, L. E. et al. 2003. J Cutan Pathol 30:459-462; Kennedy,G. A. et al. 2002. Br J Haematol 119:412-416; and Davis, T. A. 1999.Clin Cancer Res 5:611-615). The ability to detect circulating Rituximab®not only confirms the evaluation but also provides a quantitativemeasurement of the drug.

Currently, levels of circulating Rituximab® and CD20+ cells are assayedby flow cytometric, immunohistochemical and immunoassay methodsseparately. The ability to substantially simultaneously monitor bothsurface and cytoplasmic expression of these types of markers as well asthe presence of circulating drug (Rituximab) would offer an importantstep forward in assessing therapeutic effectiveness.

Another example of a therapeutic monitoring application is the treatmentof B-cell chronic lymphocytic leukemia with anti-CD52 antibody(CAMPATH-1, alemtuzumab), which was FDA, approved a few years ago. CD52is a glycophosphatidyl inositol (GPI) anchored protein that is highlyexpressed on normal lymphoid cells and monocytes as well as on a largeproportion of lymphoid cell malignancies—but not on hematopoieticprogenitor cells (Dumont, F. J. 2002. Expert Rev Anticancer Ther2:23-35). In treatment with anti-CD52 antibody, continued monitoring toevaluate remission versus tumor escape is required. After treatment,B-cells may no longer express CD52 due to B-cell depletion, tumorescape, blocking of the CD52 by the drug (CAMPATH-1) that binds thedetector antibody ((Giles, F. J., et al. 2003. Br J Haematol123:850-857). In addition the CD52 may be shed into the circulation, acharacteristic of GPI anchored proteins.

The use of a second antibody with specificity for a different epitope ofCD52 (e.g. HI186 or CF1D12) can assess tumor escape. The measurement ofCAMPATH-1 serum levels can be used to optimize dose regimens, and alsoconfirms the evaluation of tumor escape (Birhiray, R. E. et al. 2002.Leukemia 16:861-864 and Rebello, P. and G. Hale. 2002. J Immunol Methods260:285-302). The potential for anti-idiotype antibodies, though lessproblematic when a humanized monoclonal antibody, such as CAMPATH-1, isused, may also be monitored [10]. In addition, due to the toxicity ofthis treatment resulting in extensive depletion of lymphocytes, theability to quantitate differences in the level of CD52 expression mayallow stratification of responders to non-responders [11]. To monitorthis situation, analysis has historically been done separately using twoto three different technologies, such as flow cytometry for surfaceexpression and fluorescence microscopy and immunoassay for circulatingCD52 or immune-complexes.

Auto-antibodies directed against the heparin/platelet factor 4 (H:PF4)complex are responsible for heparin-induced thrombocytopenia (HIT), animportant side effect of heparin therapy (Reilly, R. F. 2003. Semin Dial16:54-60). The incidence of HIT among patients receiving heparin is 1-3%(DeBois, W. J. et al. 2003. Perfusion 18:47-53). The increased thrombingeneration is associated with decreased platelet counts (<150 k/μL) andhigh anti-heparin/PF4-antibody levels. This combination of events ispotentially life threatening. The ability to rapidly determine theamount of anti-heparin/PF4-antibody could therefore help guide clinicalmanagement. Recently a new flow cytometric assay that detects anti-H:PF4antibodies and platelet activation using Annexin V has been described(Gobbi, G. et al. 2004 Cytometry 58B:32-38. Previous to thisdevelopment, beads coated with H:PF4 have been used alone to detectanti-platelet antibodies (Tazzari, P. L. et al. 2002. Transfus Med12:193-198).

There remains a need in the art for more efficient methods of analyzingmultiple analytes in a single sample, which methods can reduce the stepsperformed on the samples, thereby improving accuracy and costeffectiveness, while preserving rare samples.

SUMMARY OF THE INVENTION

The invention provides high though-put methods for monitoring treatmentof patients being administered a soluble ligand that binds to a cellmarker. In one embodiment, the invention provides methods for monitoringtreatment of a patient in need thereof with a treatment ligand thatbinds specifically to the cell surface expressed target CD20. Theinvention CD20 monitoring method includes, in a container containing asample including bodily fluid having CD20⁺ cells obtained from thepatient, performing one of the following:

-   -   i) incubating assay components in the container with a first        soluble ligand that binds specifically to CD20 and is conjugated        to a first distinguishable fluorescent label under conditions        and for a time to allow formation of complexes of CD20 and the        first ligand; or    -   ii) adding to the container a second soluble ligand that binds        specifically to B cells and is conjugated to a second        fluorescent label under conditions and for a time to allow        formation of complexes between assay components; or    -   iii) the following combination of steps:        -   a) adding to the container a capture particle linked to CD20            antigen;        -   b) permeabilizing cells in the container; and        -   c) incubating assay components in the container with a third            soluble ligand that binds specifically to intracellular CD20            and is conjugated to a third distinguishable fluorescent            label under conditions and for a time to allow formation of            complexes of intracellular CD20 and the first ligand.            Fluorescence from the fluorescent labels in complexes formed            in the container is detected to monitor the treatment of the            patient.

In another embodiment, the invention provides methods for monitoringtreatment of a patient in need thereof with a treatment ligand thatbinds specifically to the cell surface expressed target CD20 byincubating together in a container under conditions and for a timesufficient to allow complex formation between the following assaycomponents: 1) a sample comprising bodily fluid containing CD20⁺ cellsobtained from the patient; 2) a first soluble ligand that bindsspecifically to soluble CD20 conjugated to a first distinguishablefluorescent label; 3) a second soluble ligand that binds specifically toB-cells conjugated to a second distinguishable fluorescent label; and 4)a capture particle linked to CD20 antigen. After permeabilizing cells inthe container, the assay components in the container are incubated witha third ligand that binds specifically to intracellular CD20 and isconjugated to a third distinguishable fluorescent label under conditionsand for a time to allow formation of complexes of intracellular CD20 andthe third ligand, thereby forming a mixture of components therein. Thepresence of fluorescence from the first, second or third fluorescentlabels in the mixture of complexes formed in the container is detectedto monitor the treatment of the patient.

In still another embodiment, the invention provides methods formonitoring treatment of a patient in need thereof with a treatmentligand that binds specifically to the cell surface expressed targetCD52. The CD52 treatment monitoring method includes obtaining acontainer containing a sample comprising a bodily fluid containing CD52⁺cells obtained from the patient; incubating the sample in the containerunder conditions and for a time sufficient to allow complex formationwith i) a first ligand that binds specifically to the expressed targetat the binding site of the treatment ligand conjugated to a firstdistinguishable fluorescent label and ii) one assay component selectedfrom a second ligand that binds the expressed target at a differentbinding site than the treatment ligand conjugated to a seconddistinguishable ligand; a third ligand that binds specifically to humanimmunoglobulin conjugated to a third distinguishable fluorescent label;and a first distinguishable capture particle linked to a CD52 antigen.Fluorescence from the fluorescent labels in the complexes formed in thecontainer is detected substantially simultaneously to monitor thetreatment of the patient.

In yet another embodiment, the invention provides methods for monitoringtreatment of a patient in need thereof with a treatment ligand thatbinds specifically to the cell surface expressed target CD52. The CD52treatment monitoring method can include incubating the following assaycomponents in a container under conditions and for a time sufficient toallow complex formation between: 1) a sample comprising a bodily fluidcontaining CD52⁺ cells obtained from the patient; 2) a firstdistinguishable capture particle linked to a CD52 antigen; 3) a seconddistinguishable capture particle linked to the treatment ligand.Complexes formed by this first incubation are again incubated underconditions and for a time sufficient to allow binding interaction withthe following additional assay components to form a mixture ofcomplexes: 1) a first ligand that binds the expressed target at adifferent binding site than the treatment ligand and is conjugated to afirst distinguishable fluorescent label; 2) a second ligand that bindsspecifically to the expressed target at the binding site of thetreatment ligand and is conjugated to a second distinguishablefluorescent label; and 3) a third ligand that binds specifically tohuman immunoglobulin and is conjugated to a third distinguishablefluorescent label. The presence of fluorescence from the first, secondor third fluorescent labels in the mixture of complexes formed in thecontainer is detected substantially simultaneously to monitor thetreatment of the patient.

In still another embodiment the invention provides methods formonitoring side effects of heparin therapy in a patient in need thereofthat includes incubating the following assay components in a containerunder conditions and for a time sufficient to allow complex formationbetween: 1) a sample comprising stabilized whole blood of the patient;2) a distinguishable capture particle linked to heparin:platelet factor4 complex; 3) a first soluble ligand that binds specifically to aplatelet activation antigen and is conjugated to a first fluorescentlabel; and 4) a second soluble ligand that binds specifically toplatelets and is conjugated to a second fluorescent label. Complexesformed by this first incubation are again incubated in the containerunder conditions and for a time sufficient to allow binding interactionwith a third soluble ligand that binds specifically to humanimmunoglobulins conjugated to a third fluorescent label to form amixture of complexes. Fluorescence from the first fluorescent label,second fluorescent label or the third fluorescent label in the complexesformed in the container is detected substantially simultaneously tomonitor heparin therapy in the patient.

In another embodiment, the invention provides kits for monitoringtreatment of a patient with a treatment ligand that binds specificallyto the cell surface expressed target CD20. In this embodiment, the kitincludes a first soluble ligand that binds specifically to intracellularCD20; and one or more of the following: 1) a first soluble ligand thatbinds specifically to CD20⁺ cells; 2) a second soluble ligand that bindsspecifically to CD19⁺ cells; 3) capture particle linked to CD20 antigen;and 4) one to three distinguishable fluorescent labels for conjugationto the ligands.

In yet another embodiment, the invention provides kits for monitoringtreatment of a patient with a treatment ligand that binds specificallyto CD52 antigen. This CD52 monitoring kit includes a) a firstdistinguishable capture particle linked to a CD52 antigen; and one ormore of the following: 1) a second distinguishable capture particlelinked to the treatment ligand; 2) a first soluble ligand that binds theexpressed target at a different binding site than the treatment ligand;3) a second soluble ligand that binds specifically to the expressedtarget at the binding site of the treatment ligand; 4) a third solubleligand that binds specifically to human immunoglobulin; and 5) threedistinguishable fluorescent labels for conjugation to the ligands.

In still another embodiment, the invention provides kits for monitoringheparin therapy of a patient. The invention heparin monitoring kitincludes a distinguishable capture particle linked to a heparin:plateletfactor 4 complex; and one or more of the following: 1) a first solubleligand that binds specifically to a platelet activation antigen; 2) asecond soluble ligand that binds specifically to platelets; 3) a thirdsoluble ligand that binds specifically to human immunoglobulins; and e)three distinguishable fluorescent labels for conjugation to the ligands.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting the method of the presentinvention employing “sandwich assay” steps, in which the capture mediumis coated with a ligand for the soluble target which is an antibody, asecond soluble ligand (antibody) that binds to the cellular target andwhich is associated with a fluorescent label FL1, and a third solubleligand (antibody) that binds the soluble target and is associated with asecond fluorescent label FL2. The substantially simultaneous evaluationof the complexes formed is demonstrated by a flow cytometry graph offorward light scatter vs. log of side scatter where the solid phasecapture medium can be gated separately from the cellular target(s) andtwo separate graphs of the number of fluorescent events associated withthe gated population(s) can be determined. L=lymphocytes; M=monocytes;G=granulocytes.

FIG. 2A is a schematic diagram depicting the method of the presentinvention employing “competitive inhibition assay” steps. The capturemedium is coated with soluble analyte. Also employed in the method are asoluble ligand for the cellular target that is an antibody and which isassociated with a fluorescent label FL1, and a second soluble ligand(antibody) that binds to the analyte in the sample or on the bead, andwhich is associated with a fluorescent label FL2. The potentialcomplexes formed are (1) a complex formed by the soluble ligand-FL1 andthe cellular target, (2) a complex formed by the capture medium withimmobilized soluble analyte and the second soluble ligand-FL2 (that hasnot bound to soluble analyte in the sample), and (3) the soluble analytein the sample, if any, and the second soluble ligand-FL2. Thesubstantially simultaneous evaluation of the complexes (1) and (2)formed is demonstrated by a flow cytometry graph of forward lightscatter vs. log of side scatter where the solid phase capture medium aregated separately from the cellular target(s) and two separate graphs ofthe number of fluorescent events associated with the gated population(s)are shown. The measurement of the FL2 on the capture medium-immobilizedanalyte-ligand-FL2 complex is inversely proportional to the amount ofsoluble analyte in the sample due to competition for binding between theimmobilized analyte and the soluble analyte in the sample.L=lymphocytes; M=monocytes; G=granulocytes.

FIG. 2B is schematic diagram depicting the method of the presentinvention employing alternative “competitive inhibition assay” steps. Inthis alternative, the capture medium is coated with the soluble ligandthat binds the soluble analyte. Also employed in this method is asoluble ligand for the cellular target that is an antibody and which isassociated with a fluorescent label FL1. Another component of the methodis a soluble analyte that is associated with a fluorescent label FL2.The potential complexes formed are (1) a complex formed by the solubleligand-FL1 and the cellular target, (2) a complex formed by the capturemedium with immobilized ligand for the soluble analyte and the solubleanalyte-FL2, and (3) a complex formed by the capture medium withimmobilized ligand for the soluble analyte and the soluble analyte inthe sample, if any. The substantially simultaneous evaluation of thecomplexes (1) and (2) formed is demonstrated by a flow cytometry graphof forward light scatter vs. log of side scatter where the solid phasecapture medium is gated separately from the cellular target(s) and twoseparate graphs of the number of fluorescent events associated with thegated population(s) are shown. The measurement of the FL2 on the capturemedium-immobilized ligand-bound analyte-FL2 complex is inverselyproportional to the amount of unlabeled soluble analyte in the sample.L=lymphocytes; M=monocytes; G=granulocytes.

FIG. 3 is a schematic diagram depicting the method of the presentinvention employing “immune complex” steps. The bead is coated withstreptavidin. A first ligand (antibody) to the cellular target isassociated with a fluorescent label FL1. A second ligand (antibody) tothe cellular target is associated with a fluorescent label FL2. A thirdligand (antibody associated with biotin) is targeted to the solubleanalyte. The substantially simultaneous evaluation of the complexesformed is demonstrated by a flow cytometry graph of forward lightscatter vs. log of side scatter where the solid phase capture medium isgated separately from the cellular target(s) and two separate graphs ofthe number of fluorescent events associated with the gated population(s)is shown. L=lymphocytes; M=monocytes; G=granulocytes.

FIG. 4 is a schematic diagram depicting embodiments of the inventionmethods for monitoring a patient being treated with a ligand that bindsspecifically to a cell surface expressed target CD20. An antibody thatbinds specifically to the cell surface expressed target, CD20, isconjugated to a fluorescent label FL2 (clone HRC20)-PE). A secondantibody that identifies the cell lineage (i.e. CD19) is conjugated to asecond fluorescent label FL3 (CD19-ECD®). A capture medium is covalentlylinked to CD20 antigen. A third antibody that binds specifically tointracellular CD20 is conjugated to a third fluorescent label FL1 (cloneL26-FITC). The substantially simultaneous evaluation of the complexesformed is demonstrated by a flow cytometry graph of forward lightscatter vs. log of side scatter to differentiate the capture medium fromthe cells and a side scatter vs. CD19-ECD to gate the B cells separatelyfrom the remaining cells. These graphs can then be used to provide twographs, one showing the number of events and mean intensity offluorescent label FL2 on capture beads, and the other showing thepercentages of B-cells containing CD20 on the surface (FL2) andintracellularly (FL1). L=lymphocytes; M=monocytes; G=granulocytes.

FIG. 5 is a schematic diagram depicting embodiments of the inventionmethods for monitoring of a patient being treated with a treatmentligand that binds specifically to a cell surface expressed target CD52.A first ligand (antibody) that binds specifically to expressed CD52 isconjugated to fluorescent label FL2 (CAMPATH 1G-PE) A second antibodythat binds the same expressed target antigen at a different site isconjugated to second fluorescent label FL-5 (HI186)-PC7). A thirdantibody that binds specifically to human immunoglobulin (e.g.anti-HuIgG-FITC) is conjugated to a third fluorescent label FL1. Onecapture bead is covalently linked to synthetic CAMPATH antigen having noreactivity with HI186 and the other is covalently linked to thetreatment antibody CAMPATH 1H. The substantially simultaneous evaluationof the complexes formed is demonstrated by a flow cytometry graph offorward light scatter vs. log of side scatter to differentiate the twotypes of capture medium and separate them from the cellular target. Thelymphocyte cell population can then be gated into a graph of FL 2 vs.FL5 to assess the percentage of B-cells containing both epitopes ofCD52. The CAMPATH antigen capture beads are gated into a Count vs. FL1or FL2 histogram to show the number of beads containing circulating drug(CAMPATH-1H antibody) while the CAMPATH-1H (drug) capture bead can begated into two separate histograms-one depicting the amount ofautoantibody in the plasma (Count vs. FL1) and the other depicting theamount of shed CD52 antigen in the plasma (Count vs. FL2 or FL5)L=lymphocytes; M=monocytes; G=granulocytes.

FIG. 6 is a schematic diagram depicting embodiments of the inventionmethods as used to monitor treatment of a patient with heparin therapyfor development of HIT. A bead is covalently linked to heparin:plateletfactor 4 (H:PF4) complex (Bead-H:PF4). An antibody that bindsspecifically to a platelet activation antigen (e.g. CD62p-FITC) isconjugated with a first fluorescent label FL1. A second antibody thatidentifies platelets (i.e. CD41 is conjugated with a second fluorescentlabel, FL5 (e.g., CD41-PC7). A third antibody that binds specifically toanti-H:PF4 autoantibodies (e.g. anti-HuIg-PE) is conjugated with asecond fluorescent label (FL2). The substantially simultaneousevaluation of the complexes formed is demonstrated by a flow cytometrygraph of the log of forward light scatter vs. log of side scatter todifferentiate the beads from the cells. The beads can be gated to aCount vs. FL2 histogram to measure the amount of circulatingautoantibodies. The cells from the log FS vs. log SS histogram are gatedinto a histogram depicting FL5 vs. log FS to distinguish the red cellsand white cells from the platelets. The platelets can then be sent to aFL2 vs. FL1 histogram to assess the proportion of activated plateletscontaining autoantibodies to H:PF4. Platelet concentration can bedetermined by the ratio of the red cells to the platelets or bycomparison to the bead count. (PLT=platelets; RBC=red blood cells).

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention answers the need in the art byproviding for the substantially simultaneous evaluation (detectionand/or measurement) of both soluble and bound targets in a sample. Themethod involves generally the analysis of a sample, which contains atleast one target bound to a larger structure and at least one solubleanalyte, which is unbound and free in the solution of the sample.

The general steps of the method involve adding to a single container thesample with (i) at least one soluble ligand that binds the cellulartarget, (ii) at least one soluble ligand that binds the soluble analyteor at least one competing soluble analyte that is preferably associatedwith a detectable label; and (iii) a solid phase capture medium thatbinds directly or indirectly to the soluble analyte or to the solubleligand that binds the soluble analyte. After appropriately incubatingthe sample with these additives, the sample is substantiallysimultaneously analyzed without physically separating the differentcomplexes that form within the sample. For example, one potentialcomplex forms between the cellular target and at least one solubleligand. Another potential complex forms between the capture medium bounddirectly to the soluble analyte (either labeled or unlabeled). Generallythis direct binding involves a capture medium having immobilized thereonat least one ligand (e.g., a monoclonal antibody) that binds to theanalyte. Still another complex may form between the capture medium boundindirectly to the soluble analyte. In this instance, the capture mediumhas coated thereon a ligand (e.g., biotin) that binds to another ligand(e.g., streptavidin) that is attached to a ligand for the solubleanalyte (e.g., a monoclonal antibody that binds the analyte). Anothercomplex may form between the capture medium bound to the soluble ligandthat is bound to the soluble analyte. In this instance, the capturemedium has coated thereon the soluble analyte, which binds the solubleligand for the analyte.

It should be understood that one or more of the ligands employed inthese methods are labeled with one or more detectable markers, asdescribed in more detail below. In certain competitive inhibition assayformats, one or more soluble analytes employed in these methods arelabeled with one or more detectable markers, as described in detailbelow. The lack of a physical separation step, i.e., the ability tomeasure the relevant complexes in the same container, in this methodprovides a valuable advantage in terms of efficiency and time inobtaining results of analysis, and further provides an advantage ofpreserving a small or rare sample, by using as little sample aspossible.

The Sample

Preferably the sample is a biological sample, in which the bound targetis a cell bearing at least one cellular target, and having at least onesoluble analyte. The biological sample preferably contains cells ofvarious types of biological tissue. For example, certain biologicalsamples include, without limitation, whole blood, saliva, urine,synovial fluid, bone marrow, cerebrospinal fluid, vaginal mucus,cervical mucus, sputum, semen, amniotic fluid, cell lines,cell-containing exudates, cell-containing media, cell-containing buffer,bacterial samples, viral sample, and other exudates from a patientcontaining bacteria or virus. Such samples may further be diluted withsaline, buffer or a physiologically acceptable diluent. Preferably suchdilution occurs before addition of the soluble ligand(s) or of thecompeting soluble analyte(s).

In such biological sample, the cell type bearing the cellular target maybe biological cells, particularly mammalian hematological or bloodcells, and also all vertebrate or invertebrate cells, insect cells,bacterial cells, parasites, yeast or fungal cells, algal or other plantcells, etc. Also included in this definition are viruses, virus-likeparticles, parasites, and essentially any biological colloidal particlethat has on its surface a receptor or antigen (i.e., an analyte) forwhich there exists a counter-receptor ligand or specific bindingpartner. The present invention is described specifically below usingmammalian blood cells, specifically one or more of red blood cells andwhite blood cells. The white blood cells that may be present include,without limitation, granulocytes, monocytes/macrophages, platelets,lymphocytes, lymphoblasts, blast cells, leukocytes, and dendritic cells.Other cell types of cells used in these methods include, withoutlimitation, fibroblasts, epithelial cells, epidermal cells, embryoniccells, hepatocytes, histiocytes, peritoneal cells, kidney cells, lungcells, sperm cells, oocytes, and normal and cancer cells of othermammalian tissue. The cellular target is generally, a cell surfaceantigen, an intracellular antigen, nuclear antigen, a fragment thereof,or a mixture of two or more of the preceding targets.

The soluble analyte, which is naturally occurring in such biologicalsample, or which is alternatively a competing soluble analyte employedas a component of certain embodiments of the methods of this invention,is likely to include, without limitation, a serum marker, apharmaceutical drug, a protein, a virus, a hormone, a lipid, a nucleicacid sequence, a carbohydrate, a toxin, or an antigen shed from a celltype identified above, or produced or secreted by a mammalian cell, abacterial cell, a virus, a cell infected by a virus, a cancer cell, afungus, etc., or a fragment thereof, or a mixture of two or more of thepreceding analytes.

Such naturally occurring targets and/or soluble analytes are desirableto detect or quantify due to their relationship to disease states. Thusdetection of such targets is useful in diagnosis of disease, ormonitoring of therapy, among others.

Still other types of sample which can be evaluated according to themethod of this invention include water from any source, manufacturedliquids such as gasoline, alcohol, pharmaceutical medicines, perfumes,food products, and the like. The targets and analytes in these samplesmay include adulterating compounds, such as drugs, poisons, toxins,microbial proteins and the like. Thus such targets are desirablydetected as a means of quality control for detecting unwantedcontamination or adulteration.

In certain embodiments, the sample can contain additional reagents. Forexample, where the sample is whole blood, the sample can contain ananti-coagulant, such as those described below. In another embodiment,the sample containing myeloid cells can contain an inhibitor ofphagocytosis, such as discussed below. In still another embodiment, thesample can be treated with one or more of a fixative, a phosphataseinhibitor, or a calcium inhibitor.

Ligands Useful in the Invention

Generally, the components of the method include ligands that bind eitherthe cellular target or the soluble analyte. By “ligand” is meant amoiety or binding partner that specifically binds to the target on thecell or to the soluble analyte. Such ligands are individually andindependently an antibody that binds a cellular antigen, an antibodythat binds a soluble antigen, an antigen that binds an antibody alreadybound to the cellular or soluble antigen; or fragments of suchantibodies and antigens that are capable of binding; a nucleic acidsequence sufficiently complementary to a target nucleic acid sequence ofthe cellular target or soluble analyte to bind the target or analytesequence, a nucleic acid sequence sufficiently complementary to a ligandnucleic acid sequence already bound to the cellular target or solubleanalyte, or a chemical or proteinaceous compound, such as biotin oravidin.

The ligands can be soluble or can be immobilized on the capture medium(i.e., synthetically covalently linked to a bead), as indicated by theassay format. As defined herein, ligands include various agents thatdetect and react with one or more specific cellular targets or solubleanalytes. Examples of ligands within the meaning of the presentinvention and their analytes include, without limitation, those listedin Table 1. TABLE 1 LIGAND RECEPTOR Antibody Antigen Natural ligands:cytokine or CK receptor chemokine Hormone Hormone receptor growth factorGrowth factor receptor Secondary reagents: streptavidin Biotinylatedantibody - antigen Antibody Antibody - antigen Synthetic peptidesReceptor Solubilized natural ligands: Receptor Counter- receptor CTLA-4B7 (CD80/86) Lectins (agglutinins) Complementary carbohydrate oroligosaccharide on cell-surface Glycoprotein MHC-peptide complex T cellreceptor (TCR) Oligonucleotide Complementary sequences in nucleic acids,DNA or RNA

Those of skill in the art know methods useful for construction of suchligands. All such ligands are characterized by the desired ability tobind the specified target or analyte, whether it is soluble or bound toa cell. In one preferred embodiment, the ligand of the invention is acomponent that preferentially binds to all or a portion of a cellsurface receptor. Thus, a ligand useful in this embodiment of theinvention may be an antibody or a functional fragment thereof capable ofbinding to a cell surface receptor on a WBC population. Such antibodiesor fragments include polyclonal antibodies from any native source, andnative or recombinant monoclonal antibodies of classes IgG, IgM, IgA,IgD, and IgE, hybrid derivatives, and fragments of antibodies includingFab, Fab′ and F(ab′)2, humanized or human antibodies, recombinant orsynthetic constructs containing the complementarity determining regionsof an antibody, an Fc antibody fragment thereof, a single chain Fvantibody fragment, a synthetic antibody or chimeric antibody constructwhich shares sufficient CDRs to retain functionally equivalent bindingcharacteristics of an antibody that binds a desired cell surfacereceptor, and a binding fragment produced by phage display.

Antibodies used in the examples of this invention were generallyobtained by conventional hybridoma methods and purified from ascitesfluid by ammonium sulfate (45%) precipitation, centrifugation andaffinity chromatography using protein A. The standard process of makingmonoclonal antibodies is described in G. Kohler and C. Milstein, 1975Nature, 256: 495-497. Of course, the particular method of making and thetype of monoclonal antibody is not limited to such techniques and it isenvisioned that any technique for making such antibodies is within thepractice of the invention. Any ligand that can bind cellular targets orsoluble analytes may be used, since the amplification of fluorescentintensities does not depend on the density of the particular receptorsites on a cell.

Other typical ligands can include, without limitation, a lectin, ahormone, a growth factor, or a synthetic peptide or chemical compound,or portions thereof that can bind the target or analyte. The selectionof the ligand is not a limiting factor in this invention. Exemplaryligands are illustrated in the specific embodiments of methods describedbelow and in the examples.

Detectable Labels or Markers

Where indicated, the ligands and/or the competing soluble analytesand/or the capture medium employed in the methods of this invention areassociated (for example, linked covalently) with detectable labels ordetectable markers. Detectable labels for attachment to componentsuseful in this invention may be easily selected from among numerouscompositions known and readily available to one skilled in the art ofdiagnostic assays. The reagents, ligands, competing analytes, or capturemedium of this invention are not limited by the particular detectablelabel or label system employed. In some cases, the detectable “label”can include the refractive index of a cell surface or bead.

As used herein, the terms “label” or “marker” generally refers to amolecule, preferably proteinaceous, but also a small chemical moleculethat is capable, acting alone, or in concert with other molecules orproteins, of providing a signal, that is detectable either directly orindirectly. In this invention, the marker is associated with the variousligands or competing analytes used in the assays. For example, adetectable label or marker can be a fluorescent label, a luminescentlabel, a radiolabel, or a chemiluminescent label linked (e.g,covalently) to an analyte, solid particle, cell, or ligand.

In one embodiment, preferred markers enable detection by emitting adetectable signal of a particular wavelength upon excitation by a laser.Phycobiliproteins, tandem dyes, certain fluorescent proteins, smallchemical molecules, and certain molecules detectable by other means canall be considered markers for flow cytometry analyses. See, e.g., themarkers listed in Handbook of Fluorescent Probes and Research Chemicals,6th Ed., R. P. Haugland, Molecular Probes, Inc., Eugene, Oreg. (1996).“Phycobiliproteins” are a family of macromolecules found in red algaeand blue-green algae. The biliproteins (the term “biliproteins” isequivalent to the term “phycobiliprotein”) have a molecular weight of atleast about 30,000 daltons, more usually at least about 40,000 daltons,and may be as high as 60,000 or more daltons usually not exceeding about300,000 daltons. The biliproteins will normally be comprised of from 2to 3 different subunits, where the subunits may range from about 10,000to about 60,000 molecular weight. The biliproteins are normally employedas obtained in their natural form from a wide variety of algae andcyanobacteria.

The presence of the protein in the biliproteins provides a wide range offunctional groups for conjugation to proteinaceous and non-proteinaceousmolecules. Functional groups that are present include amino, thiol, andcarboxyl. In some instances, it may be desirable to introduce functionalgroups, particularly thiol groups when the biliprotein is to beconjugated to another protein. Each phycobiliprotein molecule contains alarge number of chromophores. An exemplary ligand, e.g., an antibodymolecule directly labeled with fluorescein will have between 1 and 3chromophores associated with it. An antibody molecule (for example)directly labeled by conjugation with a phycobiliprotein may have as manyas 34 associated chromophores, each with an absorbance and quantum yieldroughly comparable to those of fluorescein.

Examples of phycobiliproteins useful in the present invention arephycocyanin, allophycocyanin (APC), allophycocyanin B, phycoerythrin(PE) and preferably R-phycoerythrin. PE is among the brightestfluorescent dyes currently available. Conjugated to an antibody, PE hasbeen used to detect interleukin-4 in a fluorescent plate assay and foundto be the only tested fluorescent label that produced adequate signal(M. C. Custer and M. T. Lotze, 1990 J. Immunol. Methods, 128, 109-117).

The tandem dyes are non-naturally occurring molecules that may be formedof a phycobiliprotein and another dye. See, for example, U.S. Pat. No.4,542,104 and U.S. Pat. No. 5,272,257. Examples of tandem dyes useful inthe present invention are phycoerythrocyanin or PC5 (PE-Cy5,phycoerythrin-cyanin 5.1; excitation, 486-580 nm, emission, 660-680 nm)[A. S. Waggoner et al, 1993 Ann. N.Y. Acad. Sci., 677:185-193 and U.S.Pat. No. 5,171,846] and ECD (phycoerythrin-texas red; excitation,486-575 nm, emission, 610-635 nm) [U.S. Pat. No. 4,542,104 and U.S. Pat.No. 5,272,257. Other known tandem dyes are PE-Cy7, APC-Cy5, and APC-Cy7μM. Roederer et al, 1996 Cytometry, 24:191-197]. Tandem dyes, PC5 andECD, have been successfully directly conjugated to monoclonal antibodiesby several methods that involve iminothiolane activation of the dye.

Preferably, the ligands and/or competing analytes and/or capture mediumof this invention are associated with, or conjugated to fluorescentdetectable fluorochromes, e.g., fluorescein isothiocyanate (FITC),phycoerythrin (PE), allophycocyanin (APC), or tandem dyes, PE-cyanin-5(PC5), PE-cyanin-7 (PC7), and PE-Texas Red (ECD). The biliproteins andtandem dyes are commercially available from various sources includingBeckman Coulter, Inc., Miami, Fla., Molecular Probes, Inc., Eugene,Oreg. and Prozyme, Inc., San Leandro, Calif. All of these fluorescentdyes are commercially available, and their uses known to the art.

Still other markers that may be directly conjugated to the components ofthe methods of this invention and used with the phycobiliproteins ortandem dyes in this invention to add additional numbers of markers(labeled ligands) to the method include small molecules that uponexcitation emit wavelengths of less than 550 nm. Such molecules do notoverlap with the emissions of the phycobiliproteins. One example of sucha marker is fluorescein isothiocyanate (FITC). Others are listed in theHandbook cited above.

Still other markers that may be employed in this method to provideadditional colors are the proteins known as the green fluorescentproteins and blue fluorescent proteins; also useful may be markers thatemit upon excitation by ultraviolet light.

A marker can be an enzyme that interacts with a substrate to produce thedetectable signal. Another marker embodiment can be a protein that isdetectable by antibody binding or by binding to a suitably labeledligand. A variety of enzyme systems operate to reveal a colorimetricsignal in an assay, e.g., glucose oxidase (which uses glucose as asubstrate) releases peroxide as a product that in the presence ofperoxidase and a hydrogen donor such as tetramethyl benzidine (TMB)produces an oxidized TMB that is seen as a blue color. Other examplesinclude horseradish peroxidase (HRP) or alkaline phosphatase (AP), andhexokinase in conjunction with glucose-6-phosphate dehydrogenase thatreacts with ATP, glucose, and NAD+ to yield, among other products, NADHthat is detected as increased absorbance at 340 nm wavelength.

Other label systems that may be utilized in the methods of thisinvention are detectable by other means, e.g., colored latexmicroparticles (Bangs Laboratories, Indiana) in which a dye is embeddedmay be used in place of enzymes to form conjugates with the inhibitorsequences or ligands and provide a visual signal indicative of thepresence of the resulting complex in applicable assays. Still otherlabel systems that may be used include nanoparticles or quantum dots.

In another embodiment such markers may preferably be reporter genes thatupon expression produce detectable gene products. Such reportersequences include without limitation, DNA sequences encoding a lux gene,beta-lactamase, a galactosidase enzyme, e.g., beta-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), a luciferase enzyme, or agluconase enzyme.

Still other suitable marker that may be attached to the components ofthe methods of this invention include membrane bound proteins including,for example, CD2, CD4, CD8, the influenza hemagglutinin protein, abiotin molecule, an avidin molecule, and others well known in the art,to which high affinity antibodies directed thereto exist or can beproduced by conventional means. Another class of markers includes fusionproteins comprising a membrane bound protein appropriately fused to anantigen tag domain from, among others, hemagglutinin or a Myc gene.Still other detectable labels may include hybridization or PCR probes.

Any number of additional, and conventionally employed, marker systemsmay be adapted to the method of this invention. One of skill understandsthat selection and/or implementation of a label system involves onlyroutine experimentation. The labels and markers discussed above may beobtained commercially from known sources.

Solid Phase Capture Medium

The solid phase capture medium is typically a capture particle, such asa physiologically compatible bead or a stabilized cellular particle,with any characteristic that allows it to be separated from the cellpopulation of the sample. Such characteristics include refractive index,size, light scatter intensity (forward, side or 90°), or carrying afluorescent detector dye to provide a unique fluorescent signature. Suchbeads suitable for use as capture particles in the invention methods areconventionally available in the art. For example, one subset of solidphase capture medium includes stable colloidal particles, such aspolystyrene beads ranging in size from between about 0.2 to about 5.0microns in diameter (i.e., colloidal-sized). Such polystyrene substratesor beads can contain aldehyde and/or sulfate functional groups, such asthe commercially available beads, e.g., from Interfacial DynamicsCorporation, Portland, Oreg.

Alternatively, the polystyrene bead has an aminodextran coating over itsperipheral surface and/or a colloidal-metal coating. Preferably anaminodextran coating is covalently bonded to the core substrate bycovalent bonds between the free amino groups of the aminodextran and theamine-reactive functional groups of the polystyrene substrate andfurther by crosslinking with an agent such as glutaraldehyde. Theaminodextran coating may generally be characterized as having a degreeof diamine substitution in the range of 1/40- 1/35 (1×-aminodextran)compared to a maximum theoretical value of 1/2.5. More preferably, thediamine substitution in the aminodextran coating is approximately 1/7 to⅛ (5×-aminodextran). Analytes, particularly protein analytes, may bereadily attached to these beads as is taught in the references citedbelow. See also, O. Siiman et al, “Covalently Bound Antibody onPolystyrene Latex Beads: Formation, Stability and Use in Analyses ofWhite Blood Cell Populations”, J Colloid Interface Sci., 233: (January2001).

A variety of aminodextran beads are described in U.S. Pat. Nos.6,074,884; 5,945,293; and 5,658,741. Aminodextran-coated monodispersedcolloidal dispersions of magnetic ferrite [U.S. Pat. No. 5,240,640],metal [U.S. Pat. No. 5,248,772], polystyrene [U.S. Pat. No. 5,466,609;U.S. Pat. No. 5,707,877; U.S. Pat. No. 5,639,620; U.S. Pat. No.5,776,706], and polystyrene-metal [U.S. Pat. No. 5,552,086; U.S. Pat.No. 5,527,713] particles may also be employed as formed bodies accordingto this invention.

Another type of bead may contain the above-described coated substratewith a layer of colloidal-sized metallic solid overlaying theaminodextran coating. Preferably this layer is uniformly dispersed overthe dispersed surface of the aminodextran layer. The colloidal metaluseful in forming the coated substrate is generally described as a metalwhich can be reduced from the ionic state to the metal(0) state by theaminodextran coating, or a metal which can form metal ions or metal ioncomplexes which have a reduction potential of about +0.7 volts orhigher. While such metal ions may include: Ag(I), Au(III), Pd(II),Pt(II), Rh(III), Ir(III), Ru(II), Os(II), the preferred metal ions forsuch use are colloidal gold(III) and colloidal silver(I). Specifically,gold/silver colloid coated polystyrene-aminodextran beads, theirpreparation, characterization and use in analyses of subpopulations ofwhite blood cells in whole blood have been described. See, e.g., U.S.Pat. No. 5,248,772; U.S. Pat. No. 5,552,086; U.S. Pat. No. 5,945,293;and O. Siiman and A. Burshteyn, 2000 J. Phys. Chem., 104:9795-9810; andO. Siiman et al, 2000 Cytometry, 41:298-307.

An alternative to this coated bead employs carboxy-functionalizedpolystyrene particles as the core substrate, coated with aminodextran byEDAC coupling as described in U.S. Pat. No. 5,639,620.

Other suitable beads that may be utilized in the methods of thisinvention are colored latex microparticles (Bangs Laboratories, Indiana)in which a dye is embedded and may be used to form complexes with thetarget, analyte or ligands. These beads also provide a visual signalindicative of the presence of the resulting complex in applicableassays. Still other suitable beads include nanocrystals, quantum dotsand similar materials.

In one embodiment the bead is from 0.05 to 20 microns in diameter. Inanother embodiment, the bead is from 5 to 7 microns. In still anotherembodiment, the capture medium is greater than 1 μM in size. Mixtures ofa variety of sizes of beads may also be employed, particularly wherethere are more than one soluble analyte to be detected. Generally, beadsize impacts the sensitivity range of the assay, because smaller beadsbind fewer antibodies (see e.g., Lindmo, cited above or Frengen citedabove). Therefore, in one embodiment, in which high sensitivity isrequired, a smaller number of larger beads is desirable for the assays.In the presence of large numbers of soluble analytes, a higher number ofbeads (both large and small) may be employed in these methods. For usein some embodiments of the present invention, the capture medium or beadis larger than the soluble analyte to be detected.

The capture medium may have bound thereto multiple ligands or multiplecompeting analytes. Each ligand bound to the capture medium is capableof binding to a soluble analyte or binding to an antibody that is itselfcapable of binding to the soluble analyte. Each competing analyte boundto the capture medium is capable of binding to a ligand (e.g., anantibody) that is capable of binding to the soluble analyte (whetherlabeled or unlabeled). Such ligands or competing analytes are associatedor immobilized on the capture medium by conventional methods. Forexample, ligands or analytes such as antibodies, antigens, or linkers(e.g. Streptavidin, Protein A) may be attached to beads depending uponformat of the analyte assay (competitive, immune-complex or sandwich) asdescribed below. The beads may also be associated with detectablelabels, preferably fluorescent labels, such as discussed above. Methodsfor attachment with such labels are disclosed in the texts cited herein.

Beads may be fluorescent or non-fluorescent, may be of different sizesor different fluorescent intensities, or both, for differentiation ofmultiple analytes. If using fluorescent intensity for labeling beads, itis preferred that the fluorescence emission should be unique for eachpopulation directed to a different analyte. Bead populations ofdifferent intensity are preferably resolvable if fluorescence of thebead is used as the only detectable label for discriminating among thesoluble analyte and cellular target. Alternatively, if the size of thebead populations is used as the sole detectable label for discriminationamong the soluble analyte and cellular target, each bead population musthave a different forward scatter (FS) or side scatter (SS) than the cellpopulation of interest in the assay.

Where the resulting analytic steps involve flow cytometry, the minimalparameters or characteristics of the beads are scatter (forward scatter(FS) and/or side scatter (SS)) or at least two fluorescent wavelengths.

The relative volumes of the bead used in the sample container of themethods described herein are dependent upon bead concentration, analytedetection limits, and the cellular target, and sample size. For example,in one embodiment about 10 μL beads may be added to per 50-100 μL bloodfor 12×75 test tubes vs. 5 μL beads for 25-50 μL blood for microplateassays.

Preferably and optionally, solutions of bead populations useful in thepresent invention include a reagent that inhibits phagocytosis of thecapture medium without damaging the target cells or inhibiting bindingthe target cells and the ligands.

Additionally or alternatively, the bead solution may contain ananti-coagulant, such as those mentioned below. Further the beadsolutions may be kept at a temperature below 37° C., and morepreferably, below 25° C., prior to addition to the sample or whenintroduced into the sample. These alternative and optional steps arealso useful for inhibiting phagocytosis of the beads when in the sample.

Assay Formats

This method may utilize any number of conventional assay formats, forexample, sandwich assays, competitive inhibition assays, immune complexassays, or others. Some of the components of these assays, as well asthe conditions under which the sample is incubated, and the inclusion ofoptional steps or reagents, are dependent upon the assay selected.However, surprisingly, these assays using both beads and cellularmarkers provide accurate results in a single analysis. There is nonegative effect on the binding of the beads in the presence of thecellular markers or vice versa. Surprisingly, there is no effect on themeasurement of light scatter of fluorescent properties of the cells inthe presence of the beads.

(1) Sandwich Assay

In one embodiment, the method can include the following steps for asandwich assay. See, e.g., FIG. 1. A sample is introduced into acontainer, such as a microtiter plate wells or test tubes. The solidphase capture medium is added to the container. In this assay, thecapture medium or bead has immobilized thereon multiple first ligandsthat are capable of binding the soluble analyte. Preferably the methodemploys mixing, and incubation at a temperature of 37° C. or lower forabout 5 minutes to up to 3 hours, and preferably for about 60 minutes.In one embodiment, the temperature is desirable lower than 25° C. or 22°C. The temperature and incubation times can be selected by one of skillin the art based upon the analyte, the analyte detection limit and theidentity of the cellular target in the sample.

Thus after incubation and occasional mixing, a “first” complex is formedin the sample which consists of the capture medium, multiple immobilizedfirst ligands, and multiple soluble analyte now bound to the capturemedium by the first ligands.

An optional washing step may be employed before addition of thefollowing components, depending upon required assay sensitivity. In someembodiments of these methods, a wash step to eliminate unbound firstligands is required for increased sensitivity.

Thereafter, suitable concentrations of at least two additional ligandsare added to the sample. One of the additional ligands is a solublesecond ligand smaller than the cellular target. The second ligand iscapable of binding to the cellular target, e.g., to a cell surface orintracellular moiety. For example, an antibody to the cell surfaceantigen is a suitable ligand here. Each second ligand is desirablyassociated with a detectable label such as described above, and multiplesecond ligands can bind to a single target cell. The other of theadditional ligands is a third ligand that is capable of binding to thesoluble analyte whether that analyte be immobilized on the capturemedium in the first complex or remaining soluble in the sample. Thisthird ligand is desirably associated with a second detectable label thatis different from the detectable label of the second ligand, i.e., theligand that binds the cellular target.

In certain embodiments of this sandwich assay method, there are morethan one second ligand directed to more than one target on the same celltype (e.g., an anti-CD45-PC5 antibody to the cell surface antigen CD45and an anti-CD 14-FITC antibody to the cell surface antigen CD14). Instill other embodiments, more than one soluble ligand is directed to thesame or to different targets on the same or different cell types. Incertain embodiments of this assay more than one ligand is employed tomore than one soluble analyte (e.g., an anti-IL-2-PE antibody to thesoluble analyte IL-2, an anti-IL-6-PC7 antibody to the soluble analyteIL-6). Alternatively, more than one soluble ligand may be used for thesame soluble analyte, or the same fluorochrome may be used for more thanone soluble analyte.

After these components are added to the sample, the sample is mixed, andincubated with occasional mixing as described above. Thus the sample nowcontains a second complex consisting of the second labeled ligand(s)bound now to the cellular target(s) and a third complex comprising thethird ligand bound to the soluble analyte which is bound through thefirst ligand to the capture medium. There may also be some small solublethird ligand-soluble analyte complexes in the sample.

Another optional step may be inserted into the assay method at thispoint, if the sample contains non-nucleated cells, such as red bloodcells, and if higher sensitivity is needed for the analysis steps below.The sample may optionally be treated with an agent to lyse thenon-nucleated cells. Another optional wash step may also be included toremove the lysed materials from the complexes or to remove excessunbound labeled ligands, depending upon required assay sensitivity.

The final step of this method is a substantially simultaneous analysisof the sample treated as described above, without physically separatingthe various complexes to be measured. Given the above steps of thismethod, one may take the sample containing these complexes anddiscriminate between the third complex comprising the third ligand boundto the soluble analyte which is bound through the first ligand to thecapture medium and the second complex consisting of the second labeledligand(s) bound now to the cellular target(s) using the same sample.Methods suitable for performing this analysis step include imageanalysis and, preferably, flow cytometric analysis. A flow cytometricanalysis is conducted by employing a gating strategy appropriate to thesample type. For example, the third complex containing the beads isgated separately from the second complex of the ligand-labeled cellsbased on light scatter and/or fluorescence intensity. Thereafter, ifmore than one fluorescent label is present on the cell target or thebead, the strategy can provide separate compensation for eachfluorescent label. Similarly other cell parameters, such asdifferentially expressed targets and intracellular targets may also bemeasured during this analysis. The amount of third complex detected isproportional to the amount of soluble analyte (unlabeled) present in thesample.

The standards for quantitation of the analyte include cell controls withserum-based analyte standards. Such standards are applicable to allassay types described herein. These standards are stabilized cells in amedia containing the soluble analytes of interest.

(2) Competitive Inhibition Assay

In still other embodiments, the method can include the following stepsfor a competitive inhibition assay. See, e.g., FIG. 2A and FIG. 2B.

In one format depicted in FIG. 2A, a sample is introduced into acontainer, such as a microtiter plate well or test tube. A knownconcentration of a first soluble ligand capable of binding to a singlecellular target is added to the sample. This first ligand is desirablyassociated with a first detectable label. Multiple of the first ligandsmay bind to the cell. At the same time a known concentration of a secondligand capable of binding the soluble analyte is added to the sample.The second ligand is associated with a second detectable label. Aftermixing and incubating for from about 5 minutes to about 3 hours,preferably up to 60 minutes, at a temperature of under 37° C., a firstcomplex is formed which includes the cellular target bound to the firstlabeled ligand and a second complex is formed comprising soluble analytebound to the second labeled ligand.

Thereafter a solid phase capture medium on which are immobilized a knownmultiple of the same analytes is added to the sample. The sample isvortexed and incubated again under the same conditions, and a thirdcomplex is formed consisting of the capture medium, the analyteimmobilized thereof and any of the second ligand in the sample that didnot bind to the soluble analyte.

An optional washing step may be employed after the addition of thecomponents, depending upon required assay sensitivity.

Another optional step may be inserted into the assay method at thispoint, if the sample contains non-nucleated cells, such as red bloodcells, and if higher sensitivity is needed for the analysis steps below.The sample may optionally be treated with an agent to lyse thenon-nucleated cells. Among such agents are included without limitation,ImmunoPrep™ reagents (Beckman Coulter), ammonium chloride, etc. Anotheroptional wash step may also be included to remove the lysed materialsfrom the complexes or to remove excess unbound labeled ligands,depending upon required assay sensitivity.

The final step of this method is a substantially simultaneous analysisof the sample treated as described above, without physically separatingthe various complexes to be measured. Given the above steps of thismethod, one may take the sample containing these complexes anddiscriminate between the first complex comprising the cellular targetbound to the first labeled ligand and the third complex consisting ofthe capture medium, the analyte immobilized thereof and any of thesecond ligand in the sample that did not bind to the soluble analyte.The amount of third complex detected is proportional to the amount ofsoluble analyte present in the sample.

In one format depicted in FIG. 2B, a sample is introduced into acontainer, such as a microtiter plate wells or test tubes. A knownconcentration of a first soluble ligand capable of binding to a singlecellular target is added to the sample. This first ligand is desirablyassociated with a first detectable label. Multiple of the first ligandsmay bind to the cell. At the same time a known concentration of acompeting soluble analyte is added to the sample. The competing solubleanalyte is preferably associated with a second detectable label. Aftermixing and incubating for from about 5 minutes to about 3 hours,preferably up to 60 minutes, at a temperature of under 37° C., a firstcomplex is formed which includes the cellular target bound to the firstlabeled ligand.

Thereafter a solid phase capture medium on which are immobilized a knownmultiple of a ligand that binds to the soluble analyte (competinganalyte or naturally occurring analyte in the sample, if any) is addedto the sample. The sample is mixed and incubated again under the sameconditions, and potential second and third complexes are formed. Asecond complex is formed by the capture medium-immobilized ligand andthe naturally occurring soluble analyte in the sample, if any(unlabeled). A third complex is formed by any of the capturemedium-immobilized ligand that did not bind to the unlabeled solubleanalyte and the competing analyte (labeled). No complex is formedbetween the competing, labeled soluble analyte and the unlabeled solubleanalyte occurring naturally in the sample.

An optional washing step may be employed after the addition of thecomponents, depending upon required assay sensitivity.

Another optional step may be inserted into the assay method at thispoint, if the sample contains non-nucleated cells, such as red bloodcells, and if higher sensitivity is needed for the analysis steps below.The sample may optionally be treated with an agent to lyse thenon-nucleated cells. Among such agents are included without limitation,ImmunoPrep™ reagents (Beckman Coulter), ammonium chloride, etc. Anotheroptional wash step may also be included to remove the lysed materialsfrom the complexes or to remove excess unbound labeled ligands,depending upon required assay sensitivity.

The final step of this method is a substantially simultaneous analysisof the sample treated as described above, without physically separatingthe various complexes to be measured. Given the above steps of thismethod, one may take the sample containing these complexes anddiscriminate between the first complex comprising the cellular targetbound to the first labeled ligand and the third complex consisting ofthe capture medium-immobilized ligand and competing analyte (labeled).Additionally the second complex of the capture medium with the unlabeledanalyte may also be detected. The amount of third complex detected isproportional to the amount of soluble analyte (unlabeled) present in thesample.

As with the sandwich assay, one may manipulate this assay formeasurement of more than one cell type, more than one cellular target ona cell type, or more than one soluble analyte by selecting from amongany number of soluble ligands, detectable labels, and solid phasecapture media on which is immobilized different ligands or competinganalytes. Methods suitable for performing the analysis step includeimage analysis and, preferably, flow cytometric analysis. A flowcytometric analysis is conducted by employing a gating strategyappropriate to the sample type. For example, the complexes containingthe beads are gated separately from the complex of the ligand-labeledcells based on light scatter and/or fluorescence intensity. Thereafter,if more than one fluorescent label is present on the cell target or thebead, the strategy can provide separate compensation for eachfluorescent label. Similarly other cell parameters, such as differentialand intracellular antigens or other targets may also be measured duringthis analysis.

The standards for quantitation of the analyte include cell controls withserum-based analyte standards. Such standards are applicable to allassay types described herein. These standards are stabilized cells in amedia containing the soluble analytes of interest.

(3) Immune Complex Assay

In one embodiment, the method can include the following steps for animmune complex assay. See, e.g., FIG. 3. A sample is introduced into acontainer, such as a microtiter plate well or test tube. A first solubleligand capable of binding to the cellular target is added to the sample.Multiple of these first ligands may bind a single target cell. Desirablythese first ligands provide a first detectable signal, preferably due toassociation with a detectable label. Also added to the sample is asecond ligand, capable of binding to the soluble analyte. This secondligand is also preferably labeled and can provide a second detectablesignal. To the same sample is added a third ligand capable of binding tothe same soluble analyte, which ligand is associated with a differentdetectable label. After mixing and incubating for from about 5 minutesto about 3 hours, preferably up to 60 minutes, at a temperature of under37° C., a first complex is formed comprising the first cellular targetand the first ligand and a second complex is formed comprising thesoluble analyte bound to one or both of the second ligand and thirdligand.

Thereafter, a solid phase capture medium on which is immobilizedmultiple fourth ligands is added to the sample. These fourth ligands arecapable of binding to the second or third ligands. After mixing, andincubating under the conditions described above, a third complex isformed. This third complex consists of the solid phase capture mediumbound to multiple fourth ligands, with each fourth ligand bound to athird ligand. Each third ligand is also bound to a soluble analyte,which is then further bound to one or more second ligands.

An optional washing step may be employed after the addition of the assaycomponents, depending upon required assay sensitivity. Another optionalstep may be inserted into the assay method at this point, if the samplecontains non-nucleated cells, such as red blood cells, and if highersensitivity is needed for the analysis steps below. The sample mayoptionally be treated with an agent to lyse the non-nucleated cells.Among such agents are included without limitation, ImmunoPrep reagents(Beckman Coulter), ammonium chloride, etc. Another optional wash stepmay also be included to remove the lysed materials from the complexes orto remove excess unbound labeled ligands, depending upon required assaysensitivity.

The final step of this method is a substantially simultaneous analysisof the sample treated as described above, without physically separatingthe various complexes to be measured. Given the above steps of thismethod, one may take the sample containing these complexes anddiscriminate between the first complex, the second complex and the thirdcomplex. The amount of third complex detected is proportional to theamount of soluble analyte (unlabeled) present in the sample.

As with the sandwich assay, one may manipulate this assay formeasurement of more than one cell type, more than one cellular target ona cell type, or more than one soluble analyte by selecting from amongany number of soluble ligands, detectable labels, and solid phasecapture media on which is immobilized different analytes. Methodssuitable for performing the analysis step include image analysis and,preferably, flow cytometric analysis. A flow cytometric analysis isconducted by employing a gating strategy appropriate to the sample type.For example, the complex containing the beads is gated separately fromthe complex of the ligand-labeled cells based on light scatter andfluorescence intensity. Thereafter, if more than one fluorescent labelis present on the cell target or the bead, the strategy can provideseparate compensation for each fluorescent label. Similarly other cellparameters, such as differential and intracellular targets or antigensmay also be measured during this analysis.

The standards for quantitation of the analyte include cell controls withserum-based analyte standards. Such standards are applicable to allassay types described herein. These standards are stabilized cells in amedia containing the soluble analytes of interest.

Optional Method Steps

The methods of this invention can also include a number of optionalsteps.

(1) Washing Steps

For example, where increased sensitivity of the assays are desirable,washing steps with buffer, or diluent can be introduced into themethods. Generally, such washing steps can be introduced after theincubation of the sample with the capture medium to eliminate materialsnot bound to the capture medium. Alternatively, such washing steps canfollow incubation with soluble ligand to eliminate uncomplexedmaterials. Still another option includes washing the sample after anoptional lysis step to rid the sample of lysed red blood cellcomponents.

(2) Inhibiting Phagocytosis

Another optional step suitable for the methods of this invention is theaddition of a reagent that inhibits phagocytosis of the capture mediumby cells, particularly myeloid cells in the sample, without damaging thetarget cells or inhibiting binding the target cells and the ligands usedin the methods. A suitable phagocytosis inhibitor is sodium azide(preferably, at a concentration of less than 0.01% v/v). Gliotoxin,gliotoxin-trisulfide and gliotoxin-tetrasulfide and related compoundsbelonging to the class of epipolythiodioxopiperazines also inhibitphagocytosis by macrophages, white cells that participate in the host'sdefense system. See, e.g., U.S. Pat. No. 4,886,796. Another suitablephagocytosis inhibitor is cytochalasin B (see also, U.S. Pat. No.5,162,990). Other phagocytosis inhibitors include protein kinaseinhibitors, an excess of heavy metals such as zinc, cadmium, lead,mercury, etc., phosphatase inhibitors such as pyrophosphate andlevamisole, an excess of adenosine or the polyamines putrescine andspermidine, cycloheximide, EDTA, bromoenol lactone, and otherphospholipase inhibitors and cytochalasin D.

The phagocytosis inhibitors may be added to the bead solutionsparticularly when the biological samples contain myeloid cells, becausephagocytosis of beads by myeloid cells is common. While thephagocytosis-inhibiting reagent may be added to the capture medium priorto addition of the capture medium to the sample, it is also possible toadd the phagocytosis-inhibiting reagent to the capture medium at thesame time the capture medium is added to the sample. Alternatively, thephagocytosis-inhibiting reagent is added to the sample prior to additionof the capture medium to the sample. The phagocytosis-inhibiting reagentis added to the sample at the same time the capture medium is added tothe sample. In one embodiment, it has been determined that where themethod employs beads 1 μm or less in diameter, it is preferable tointroduce multiple phagocytic inhibitors, such as combinations of theinhibitors identified above.

(3) Lysis

Another optional step of the method for samples that contain blood cellsincludes lysing the sample to remove the generally very numerousnon-nucleated blood cells, including red blood cells prior to theanalyzing step. Lysing agents, preferably detergents, more preferablynonionic detergents, are used to break down cell membranes, thusreleasing DNA, RNA and proteins from the cells. Any suitable lysingagent may be employed. Buffered halides, such as ammonium chloride andTrizma based (e.g., about 7.5 g ammonium chloride and 2 g Tris perliter), define one suitable class of lysing agents, where the undesiredcells include red blood cells. Optionally, before the lysing, the cellsare subjected to a preliminary fixing step, such as by contacting themwith a suitable fixing agent, heating them or both. For instance, thecells are contacted with a buffered antimicrobial saline solutionincluding a suitable amount of a fixative (e.g., about 0.11%formaldehyde).

Still other lytic agents are included without limitation, ImmunoPrep™reagents (Beckman Coulter), ammonium chloride, etc. In one embodiment, alytic reagent is Bacterial Protein Extraction Reagent (BPER), aproprietary mixture of nonionic detergents marketed by the PierceChemical Company. Other nonionic detergents are useful and manydetergents are operable, even some anionic and cationic detergents undercertain applications. The nonionic detergent lysing agents are generallybe added to the sample in a concentration of about 0.1 to 5, morepreferably 0.5 to 2 wt %. Other known lysing agents can also be usedwith the technology such as freeze/thawing, French cell press, enzymes,microfluidization, sonication, etc.

As stated above, the sample may then be optionally washed after lysis.

(4) Adding an Inhibitor of Cellular Activation

Still another optional step that can be included in the methodsdescribed herein includes contacting the sample with an inhibitor ofcellular activation. The inhibitor of cellular activation is contactedwith the sample prior to or substantially simultaneously with theaddition to the sample of the capture medium or ligands used in themethods. The inhibitor of cellular activation can be one or more of ananticoagulant, an inhibiting reagent, a fixative or an inhibitingreaction temperature.

(a) Anticoagulants

Anticoagulation of the sample can be accomplished by binding orchelation of calcium ions by a variety of substances. Conventionalanticoagulants include, without limitation, ethylenediaminetetraaceticacid (EDTA) or a salt thereof, a citrate salt of sodium or potassium, anoxalate salt of sodium or potassium, or combinations thereof. Othertraditional anticoagulants include natural enzymatic inhibitors of thecoagulation sequence, such as heparin or sodium fluoride or hirudin.Still other anticoagulants include, without limitation, protease,protein kinase inhibitors such as phenylmethylsulfonylfluoride (PMSF),4-(2-aminoethyl) benzenesulfonyl-fluoride (AEBSF), tosyl-lysinechloro-methyl ketone (TLCK), tosyl-phenylalanine chloromethyl ketone(TPCK), leupeptin, epstatin A, 1-(5-isoquinolinesulfonyl) piperazine.Such anticoagulants or preservatives may be used alone or in combinationfor addition to the sample. See, for example, U.S. Pat. Nos. 5,935,857and 4,528,274. Anticoagulants may be added to the sample in thisinvention preferably prior to the addition of the ligands and/or capturemedium.

(b) Fixatives

Another optional step to be added to the methods above includes theaddition of a fixative to the sample prior to the introduction of theligands or capture medium. “Fixatives” include formaldehyde,paraformaldehyde, and glutaraldehyde, dehydrating alcohols, glyoxal, andorganic acids, such as acetic acid, formic acid, and picric acid,mercuric compounds, tannic acid and many other compounds. Another usefulfixative is described in U.S. Pat. No. 5,459,073 which fixative has lowtoxicity employing a formaldehyde donor, such as diazolidinyl urea,imidazolidinyl urea, dimethylol-5,5-dimethylhydantoin, dimethylol ureaand the like rather than formaldehyde itself.

(c) Inhibiting Reagents

Still another optional step is to add to the sample an inhibitingreagent to control cellular activation. Suitable reagent compositionscan include one or more protease inhibitor(s). A non-exclusive list ofprotease inhibitors for use in the present invention includes the serineprotease inhibitors, such as 4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride (AEBSF), which has a molecular weight of 230.7 andinhibits catalytic activity of the protease active site; antithrombinplasma protein (60,000 MW) that inhibits thrombin and other serineproteases in the blood clotting cascade; or4-amidinophenylmethanesulfonyl-fluoride-HCl (APMSF, 352.7 MW), anirreversible inhibitor of trypsin-like serine proteases. Still otherserine proteases include Aprotinin (6500 MW) that inhibits serineproteases by tightly binding to the active site of the enzyme;diisopropyl phosphorofluoridate (DFP, 184.2 MW), a very toxic,irreversible inhibitors of serine proteases and acetylcholine esterase;phenylmethanesulfonyl fluoride (PMSF, 174.2 MW), which is another toxic,irreversible inhibitor that acts by chemically modifying the active siteof the enzyme; and α-toluenesulfonyl fluoride.

Other suitable serine and cysteine protease inhibitors useful in themethods of this invention include antipain (678.2 MW), a reversibleinhibitor of proteases and of RNA synthesis; chymostatin (600 MW), areversible inhibitor of some serine and cysteine proteases; leupeptin(475.6 MW) a reversible competitive inhibitor of trypsin-like proteases;L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanone-HCl (TLCK, 369.3 MW),which inhibits irreversibly by chemically altering the enzyme activesite; and L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone (TPCK, 351.8),which irreversibly inhibits by chemically altering the enzyme activesite.

Still other suitable cysteine protease inhibitors useful in thisinvention include E-64 (357.4 MW), a non-competitive irreversibleinhibitor of cystein proteases. Other suitable protease inhibitorsinhibit metalloproteases. For example, amastatin (511 MW) is a non-toxicreversible inhibitor; bestsatin (244.8 MW) is a multi-functionmetallo-protease inhibitor that has anticarcinogenic andimmunomodulating properties; diprotin (341.5 MW), a reversibleinhibitor; EDTA (372.3 MW) a reversible inhibitor that acts by chelatingenzyme cofactors and may interfere with other metal dependent biologicalprocesses. Other metaloprotease inhibitors include vanadium, molybdatesalts, and 1,10-phenanthroline. Still other suitable inhibitors for usein this invention are aspartic protease inhibitors, such as pepstatin(685.9 MW) a peptide that provides reversible inhibition.

In one embodiment, the methods above include the step of introducinginto the sample a single inhibitor. In another embodiment, the inventionincludes adding combinations of two or more such inhibitors, to permituse of small amounts of those inhibitors that are toxic or causeotherwise undesirable effects if used alone in large concentrations. Itis desirable for the concentration of protease inhibitor(s) in thestabilizing reagent composition to be up to about 10 mM. However, therange of concentrations is entirely dependent upon the inhibitor(s)used. This range is determined based upon the experimental data ofinhibition of platelet activation, as described herein. One of skill inthe art given the teachings provided herein would readily be able todetermine, with only a minimal and conventional amount ofexperimentation, a desirable concentration for each specific inhibitorused in the assay methods.

Another group of useful inhibitors includes one or more phosphataseinhibitor(s). A non-exclusive list of suitable phosphatase inhibitorsincludes, without limitation, pyrophosphate, microcystin 1A, microcystin1R, tetramisole, 1-4-bromotetramisole, tautomycin, okadaic acid,calyculin, thrysiferyl-23-acetate, cantharidine, vanadium salts, sodiumorthovanadate, tartrate salts, phloridzin, molybdate salts, andimidazole. For other suitable inhibitors, see Handbook of EnzymeInhibitors, Melmward Sollner (1989), ISBN 3-527-26994-0; ISBN0-89537-860-0, incorporated by reference herein. In one embodiment, themethods of this invention include adding a single phosphatase inhibitorto the sample.

In another embodiment, the methods of this invention include addingcombinations of two or more such inhibitors, to permit use of smallamounts of those inhibitors that are toxic or cause otherwiseundesirable effects if used alone in large concentrations. It isdesirable for the concentration of a phosphatase inhibitor(s) in thesample to be up to about 120 mM. However, the range of concentrations isentirely dependent upon the inhibitor(s) used. This range is determinedbased upon the experimental data of inhibition of platelet activation,as described herein. One of skill in the art given the teachingsprovided herein would readily be able to determine, with only a minimaland conventional amount of experimentation, a desirable concentrationfor each sample.

(d) Temperature

Another optional step of the present invention useful for inhibitingcellular activation and making the processes of this invention moreefficient is the use of inhibiting reaction temperatures in the methodof below 25° C. Preferably, such lower temperature incubations can occurat a temperature of between 4° C. and 25° C. In one embodiment, thetemperature is below 20° C. In another embodiment, the temperature isbelow 15° C. In still another embodiment, the temperature is below 10°C. In still another embodiment, the temperature is below 7° C. One ofskill in the art given the disclosures herein may readily select theappropriate temperature for the method employed.

Specific Methods of the Invention

The methods of the present invention are useful in diagnosis of avariety of mammalian diseases or conditions. Examples of such diseasesor conditions include, without limitation, sepsis, inflammation,autoimmune disease, cardiovascular disease, viral infection, bacterialinfection, cancer, and drug activities, half-life, or interactions.Exemplary drugs include insulin, biological agents (e.g. Rituximab®) andchemotherapeutics. The methods of the present invention are also usefulfor evaluation of food or water or other products for contamination withmicroorganisms or toxins or other contaminants.

In one embodiment, the above described assay methods of this inventionare useful in a method for diagnosing sepsis or monitoring the progressthereof. This method is accomplished by performing the desired assaymethod above with soluble ligands that bind cellular targets including,but not limited to CD64 (N), HLA-DR (Mo), CD11a, CD14 (or CD64)/CD16(Mo), CD16 (N) and CD 142 (tissue factor) and using soluble ligands andcapture medium that bind directly or indirectly the soluble analyte,which may be one or more of IL-6, IL-10, IL-1, TNF-α, neopterin,C-reactive protein, procalcitonin, or activated Protein C.

In another embodiment, the methods above may be adapted for use indiagnosing autoimmune disease or monitoring the progress thereof.According to this aspect of the invention, the assay methods aboveemploy ligands that bind one or more of the cell types includingactivated T cells and activated B cells by one or more of the cellsurface or intracellular antigens that characterize those cells. Themethods also use the ligands and capture medium to bind a solubleanalyte, which may be one or more of C-reactive protein, anautoantibody, a chemokine, or a cytokine. The selection of chemokine orcytokine used as the soluble reagent may be readily made by one of skillin the art.

In still a further embodiment, the methods of this invention are usefulin diagnosing cardiovascular disease or monitoring the progress thereof.Such methods employ as the cellular target one or more ofplatelet-leucocyte aggregates, or CD 142 (TF) and use ligands that bindthereto. This method is useful in also targeting the soluble analyte,which may be hsC-reactive protein, troponin, or myoglobin. Suitableligands and capture medium for use in this method may be designed andselected by one of skill in the art given this disclosure.

A method for differential diagnosis of viral and bacterial infections orfor use in monitoring the progress thereof employs the assay stepsdisclosed herein with ligands capable of binding a cellular target,which includes, without limitation, one or more of HLA-DR, CD4/CD8,CD38, CD64(N), or CD14 (or CD64)/CD16 (Mo), CD16 (N). The solubleanalyte, which may be one or more of IFNγ, neopterin, or C-reactiveprotein is detected by ligands and capture medium that bind directly orindirectly these analytes.

In still another embodiment, the methods of the invention are suitablefor detecting and monitoring contaminants in fluid, such as watersystems, or other liquid products. For example, water may be examinedfor the presence of bacterial cells by using ligands to cell surfaceantigens or intracellular antigens of bacterial origin, and for solubleanalytes, such as toxins, by using a capture medium on which isassociated a legend to the toxin. For example, in one embodiment, thesoluble analyte is an enterotoxin, such as cholera, and the cellulartarget is the enterococcus. One of skill in the art may select otherexamples of such pollutants and targets for suitable use in methods ofthis invention.

The methods and compositions of this invention are also adaptable to thediagnosis and monitoring of other diseases and conditions, based on theidentification of cellular targets, soluble targets and ligands thatbind thereto, as directed by this specification.

Accordingly, in yet further embodiments illustrated in FIG. 4, theinvention provides methods for monitoring treatment of a patient in needthereof being treated with a ligand that binds specifically to the cellsurface expressed target CD20. The CD20 monitoring method has variousembodiments depending on the breadth of analysis desired. In thesimplest format, the method includes obtaining a container containing asample comprising bodily fluid containing CD20⁺ cells obtained from thepatient and adding to the container under conditions and for a time toallow formation of complexes between assay components one or both of thefollowing: 1) permeabilizing cells in the container; and incubatingassay components in the container with a first soluble ligand that bindsspecifically to intracellular CD20 and is conjugated to a firstdistinguishable fluorescent label under conditions and for a time toallow formation of complexes of intracellular CD20 and the first ligand;or 2) adding to the container a second soluble ligand that bindsspecifically to B cells and is conjugated to a second fluorescent labelunder such conditions. The presence of fluorescence from fluorescentlabels in complexes formed in the container is then detected to monitorthe treatment of the patient. For example, the intensity of fluorescencefrom only the first label indicates the amount of intracellular CD20 onthe B-cells in the sample. In the second instance, the intensity offluorescence from the second fluorescent label indicates the amount ofall B cells in the sample.

Using an imaging analyzer or flow cytometer, the relative intensities ofthe fluorescence from the fluorescent labels in complexes formed in thecontainer can be substantially simultaneously detected to obtain furtherinformation about the patient's response to treatment. For example, whenthe first alternative is selected, the assay may further include addingto the container one or both of the second soluble ligand and/or a thirdsoluble ligand that binds specifically to CD20⁺ cells. Fluorescence fromcomplexes formed in the container can be analyzed to determine thepercentages of B-cells containing CD20 on the surface (FL2) andintracellularly (FL2 and FL1), respectively, in FIG. 4. In yet anotherexample, the method further comprises adding a third ligand that bindsspecifically to a capture particle in the incubation and detecting therelative intensity of the third fluorescent label to determine therelative amount of circulating treatment ligand present in the patient.

In yet another embodiment, the invention method for monitoring CD20treatment includes incubating together in a container, such as analysistube, under conditions and for a time sufficient to allow complexformation between the following assay components: 1) a sample comprisingB-cells from blood or bone marrow of the patient; 2) a first ligand thatbinds specifically to soluble CD20 conjugated to a first distinguishablefluorescent label; 3) a second ligand that binds specifically to B-cellsconjugated to a second distinguishable fluorescent label; and 4) acapture particle covalently linked to CD20 antigen. Unbound first andsecond ligands and unbound components of blood plasma are optionallyremoved from the container and cells remaining therein arepermeabilized. A third ligand that binds specifically to intracellularCD20 conjugated to a third distinguishable fluorescent label isincubated in the container with the complexes formed during the firstincubation under conditions and for a time to allow binding betweenintracellular CD20 and the third ligand. Presence of fluorescence fromone or more of the first, second and third fluorescent labels bound tothe capture bead and to cells in the sample is substantiallysimultaneously detected to monitor the treatment of the patient. Ligandsthat bind specifically to B-cells in the assay methods are selected tobind to a cell surface marker of B-cells in general, such as CD19, CD5,CD22, CD24 and the like.

The sample used in the assay methods may be contained in or obtainedfrom whole blood or bone marrow, in which case, the method may furthercomprise lysing cells in the container prior to introduction of thethird ligand, for example, for binding to intracellular CD20. Relativeintensities of the first, second and third fluorescent labels bound tocells and/or to the capture particle can be detected substantiallysimultaneously by image analyzer or flow cytometry with withoutseparating the complexes formed during the assay prior to the detection.

Due to the nature of the complexes that can form in the container as aresult of the invention CD20 monitoring method, valuable information canbe learned concerning the success of the patient's treatment. Forexample, detection of the relative intensity of the first fluorescentlabel attached to the capture particle can be used to determine therelative amount of circulating treatment ligand present in the blood ofthe patient. On the other hand, detected intensities of the third and/orsecond fluorescent labels is directly proportional to the degree ofdepletion of B-cells in the patient. In addition, the detection of thefirst fluorescent label is low as compared with detection of the secondand third fluorescent labels a blockage of CD20 on B-cells of thepatient is indicated.

A more detailed embodiment of the method for monitoring CD20 treatmentprovides additional information regarding the condition of the patientand includes incubating together in a container under conditions and fora time sufficient to allow complex formation between the following assaycomponents: a sample comprising blood or bone marrow obtained from thepatient; a first ligand that binds specifically to CD20+ cellsconjugated to a first distinguishable fluorescent label; a second ligandthat binds specifically to B-cells conjugated to a seconddistinguishable fluorescent label; and a capture particle covalentlylinked to CD20 antigen. After the incubation, cells in the container arepermeabilized and assay components are incubated again with a thirdligand that binds specifically to intracellular CD20 conjugated to athird distinguishable fluorescent label under conditions and for a timeto allow binding between intracellular CD20 and the third ligand.Fluorescence from the first, second or third fluorescent labels incomplexes formed in the container is detected to monitor the treatmentof the patient, as described above.

In one aspect, the ligands used in the invention methods for monitoringpatient treatments can be antibodies, preferably monoclonal antibodies,and the treatment ligand is an antibody approved by the FDA foradministration to patients in treatment of a disease associated withexpression of CD20. For example, Rituximab® and Bexxar™ monoclonalantibodies are currently approved by the FDA for administration topatients in treatment of B-cell lymphoma and either can be used as thetreatment ligand in the invention method to monitor the course of suchtreatment.

In still another embodiment, the invention provides methods formonitoring treatment of a patient in need thereof being treated with atreatment ligand that binds specifically to the cell surface expressedtarget CD52. The invention CD52 monitoring method comprises incubatingin a container, such as an analysis tube, under conditions and for atime sufficient to allow complex formation between a sample from thepatient comprising a bodily fluid containing B-cells, such as from bloodor bone marrow, and a treatment ligand that binds specifically to thecell surface expressed target CD52. The invention CD52 monitoring methodincludes, incubating a sample comprising a bodily fluid containing CD52⁺cells obtained from the patient in a container under conditions and fora time sufficient to allow complex formation together with a firstdistinguishable capture particle covalently linked to a CD52 antigen(for binding to circulating drug) and/or a second distinguishablecapture particle covalently linked to the treatment ligand (to detectautoantibody formation and any shed CD52 antigen). One of the followingassay components is added to the container for the incubation: a firstligand that binds specifically to the expressed target at the bindingsite of the treatment ligand conjugated to a first distinguishablefluorescent label; a second ligand that binds the expressed target at adifferent binding site than the treatment ligand conjugated to a seconddistinguishable ligand; a third ligand that binds specifically to humanimmunoglobulin conjugated to a third distinguishable fluorescent label.Fluorescence from the fluorescent labels in the complexes formed in thecontainer is detected substantially simultaneously to monitor thetreatment of the patient. Optionally, a fourth ligand that bindsspecifically to B-cells can also introduced for incubation with thesample, and the method further comprises determining the relativeintensity of the fourth fluorescent label.

If the sample comprises blood, the method may further comprise removinguncomplexed ligand-fluorescent label conjugates and uncomplexed plasmacomponents from the container prior to the incubation or detection stepsof the method. Similarly, if the sample comprises whole blood, red bloodcells can be removed from the container or lysed prior to incubation ordetection steps of the method. Additionally, if the sample compriseswhole blood, the method can further comprise stabilizing the sampleprior to b) to prevent artifactual activation, using a procedure knownin the art or as illustrated in the Examples herein.

Alternative embodiments of the invention methods for monitoringtreatment of a patient being treated with a CD52 ligand can involveaddition of further ligands to the incubation and detection steps, asillustrated in FIG. 5. In one example, wherein the first ligand isselected for incubation with the sample in the container, the method canfurther include adding to the container after the incubation either thefirst capture particle or a second distinguishable capture particlelinked to the treatment ligand, for example, covalently. Alternatively,the third ligand can be added to the container and incubated with eitherone or both of the first capture particle and the second captureparticle. In another example, wherein the second ligand is selected foraddition to the container and incubation with the sample, the methodfurther includes adding to the container for incubation with the sample,one or both of the first capture particle and a second distinguishablecapture particle linked to the treatment ligand, for example covalently.

In one embodiment, the invention method for monitoring treatment of apatient with a treatment ligand that binds specifically to the cellsurface expressed target CD52 can comprise:

-   -   a) incubating the following assay components in a container        under conditions and for a time sufficient to allow complex        formation between:        -   1) a sample comprising blood or bone marrow obtained from            the patient;        -   2) a first distinguishable capture particle linked to a CD52            antigen; and        -   3) a second distinguishable capture particle linked to the            treatment ligand;    -   b) incubating in the container the complexes formed in a) under        conditions and for a time sufficient to allow binding        interaction with the following additional assay components;        -   1) a first ligand that binds the expressed target at a            different binding site than the treatment ligand, and which            is conjugated a first distinguishable fluorescent label;        -   2) a second ligand that binds specifically to the expressed            target at the binding site of the treatment ligand            conjugated to a second distinguishable fluorescent label;            and        -   3) a third ligand that binds specifically to human            immunoglobulin conjugated to a third distinguishable            fluorescent label; and    -   c) detecting substantially simultaneously fluorescence from at        least one of the first, second or third fluorescent labels in        complexes formed in the container to monitor the treatment of        the patient.

As in other embodiments of the invention methods, the detecting can beaccomplished using an image processor or flow cytometer to determine therelative intensities of two or more of the fluorescent labels in thecontainer. Due to the nature of the complexes that can form in thecontainer as a result of the invention CD52 monitoring methods, valuableinformation can be learned concerning the success of the patient'streatment. For example, quantitation of the relative intensities of thethird fluorescent label or the second fluorescent label in the samplecan be used to determine the relative amount of circulating treatmentligand in the patient. In another example, detection of the relativeintensities of the first fluorescent label or the second fluorescentlabel is useful in determining the relative amount of shed CD52 antigenin the blood of the patient. In still another illustration, detectingthe relative intensity of the third fluorescent label can be used todetermine the relative amount of circulating autoantibody to the drug orthe amount of circulating drug in the patient. The invention methods canalso be used to determine the relative amount of CD52 present on thesurface of B-cells even when the drug may be masking the CAMPATHepitope.

The measurement of serum levels of treatment ligand (e.g., CAMPATH-1serum levels) can be used to optimize dose regimens, and also willconfirm the evaluation of tumor escape [8,9]. The potential foranti-idiotype antibodies, though less problematic with humanizedmonoclonal antibodies such as CAMPATH-1H, may also be monitored. Inaddition, due to the toxicity of this treatment (e.g., extensivedepletion of lymphocytes), the ability to quantitate differences in thelevel of CD52 expression may allow stratification of responders tonon-responders.

In one aspect, in the invention methods for monitoring anti-CD52treatment, the ligands can be antibodies, preferably monoclonalantibodies and the treatment ligand is one approved by the FDA foradministration to patients in treatment of a disease associated withexpression of CD52. For example, CAMPATH-1H monoclonal antibody iscurrently approved by the FDA for administration to patients intreatment of B-cell chronic lymphocytic leukemia and can be used as thetreatment ligand in the invention method to monitor the course of suchtreatment. In such a case a monoclonal antibody that does not bind tothe CAMPATH-1 epitope of CD52, such as CD52 antibody clone HI186, canconveniently be used as the second ligand in the assay, although anyligand that binds to an epitope to which CAMPATH-1H does not bind canalso be used for this purpose. The synthetic antigen attached to thefirst capture bead may also be selected to have no reactivity with theepitope of CD52 to which the second ligand binds.

In still another embodiment the invention provides methods formonitoring side effects of heparin therapy in a patient, such asheparin-induced thrombocytopenia (HIT). The invention method includesincubating in a container under conditions and for a time sufficient toallow complex formation between the following assay components: 1) asample comprising stabilized whole blood of the patient; 2) a firstdistinguishable capture particle linked to a heparin:platelet factor 4(H:PF4) complex; 3) a first ligand that binds specifically to a plateletactivation antigen conjugated to a first fluorescent label, and 4) asecond ligand that binds specifically to all platelets and is conjugatedto a second fluorescent label. After the incubation, unbound first andsecond ligand and unbound plasma components are optionally removed fromthe container. Then a third ligand that binds specifically to humanimmunoglobulin conjugated to a third fluorescent label is added tocontents of the container and the contents are incubated underconditions and for a time sufficient to allow complex formationtherebetween. The presence of the first fluorescent label, secondfluorescent label and third fluorescent label in complexes formed in thecontainer is detected to monitor heparin therapy of the patient. Thedetection can include using an image analyzer or flow cytometer todetect the percentage of first and second fluorescent labels in thecomplexes to determine the percent of platelet activation antigen in theblood of the patient. Alternatively, the detecting can involve detectingthe mean fluorescence intensity of the third fluorescent label bound tothe capture particle to assess the amount of anti-heparin autoantibodyin the blood of the patient. Alternatively, the detecting step caninclude detecting the ratio of red cells to platelets in the sample todetermine platelet concentration in the sample.

A useful monoclonal antibody for use as the first ligand is ananti-CD62p antibody. CD62p antibody is used to target plateletactivation, and -CD41 conjugated to a second distinguishable fluorescentlabel can be used as a gating reagent for platelets.

These methods, which allow for substantially simultaneous analysis ofrelevant cellular and soluble targets, cellular antigens, cellcharacteristics and hematology parameters, provide a more completepicture than do prior art methods of a patient's medical status withregard to both cellular and soluble mediators, activators or inhibitors.This invention permits multiple assays to be conducted in a single testtube or microtiter plate and allows a comprehensive snapshot of patientstatus or drug effects. The advantages of the methods of this inventioninclude decreased sample size (e.g., blood) requirements, which areparticularly important for pediatric and geriatric patients, increasedaccuracy or clinical monitoring, increased throughput efficiency,reduced time and labor to conduct the tests, and decreased overall costto a patient. For example, the ability to assess activated immune cellsin combination with a variety of soluble analytes can improve both thediagnosis and monitoring of the above-noted diseases.

If the sample comprises blood, the method may further comprise removinguncomplexed ligand-fluorescent label conjugates and uncomplexed plasmacomponents from the container prior to the incubation or detection stepsof the method. Additionally, the method can further comprise stabilizingthe sample prior to b) to prevent artifactual activation, using aprocedure known in the art or as illustrated in the Examples herein.

Kits

For convenience, the conventional reagents for high throughput assays orother diagnostic assays useful according to this invention may beprovided in the form of kits. In yet another aspect of this invention, akit is provided for performance of the above-described methods.Preferably such kits are employed for performing the diagnostic methodsof this invention and/or monitoring therapy. However, such kits can beassembled for research purposes also. Thus, a kit of the presentinvention desirably contains the components taught above, e.g., at leastone soluble ligand that binds a cellular target in the sample; at leastone soluble ligand that binds a soluble analyte in the sample or atleast one competing soluble analyte (preferably labeled); and a solidphase capture medium that binds directly to the soluble analyte,indirectly to the soluble analyte, or to the soluble ligand that bindsto the soluble analyte. The kits also include instructions forperforming the particular assay, various diluents and buffers, andsignal-generating reagents, such as fluorescent labels, enzymesubstrates, cofactors and chromogens. Other components may includeindicator charts for calorimetric comparisons, disposable gloves,decontamination instructions, applicator sticks or containers, and asample preparatory cup.

In one embodiment of the present invention, a kit useful for theperformance of the above-described sandwich assay includes, as acomponent, a solid phase capture medium associated with multiple firstligands that bind the soluble analyte. Another kit component is thesoluble ligand that binds the cellular target and is associated with afirst detectable label. The kit further comprises a third ligand that iscapable of binding to the soluble analyte-first ligand-capture mediumcomplex. The third ligand associated with a second detectable label.

In another embodiment, a kit for performing one of the competitiveinhibition assays described above, contains a first ligand associatedwith a first label. Multiple of the first ligands are capable of bindingto a single cellular target. Another component is a second ligandassociated with a second label. The second ligand is capable of bindinga soluble analyte. Still another component is the solid phase capturemedium associated with multiple of the soluble analytes immobilizedthereon.

In another embodiment, a kit for performing another of the competitiveinhibition assays described above, contains a first ligand associatedwith a first label. Multiple of the first ligands are capable of bindingto a single cellular target. Another component is a competing analyteassociated with a second label. Still another component is the solidphase capture medium on which are immobilized multiple of ligandscapable of binding to the soluble analyte (either competing solubleanalyte or soluble analyte naturally occurring in the sample).

In yet another embodiment, a kit for performing the immune complex assayof this invention includes a first ligand capable of binding to a firstcellular target and providing a first detectable signal; a second ligandcapable of binding to the soluble analyte and providing a seconddetectable signal; a third ligand capable of binding to the same solubleanalyte; a solid phase capture medium on which is immobilized multiplefourth ligands, the fourth ligands capable of binding to the thirdligands.

Such kits are useful for evaluating blood samples for purposes ofdetermining disease states associated with inappropriate types ornumbers of blood cells, blood cell types or bound components or solubleantigens or analytes thereof. Thus, such a kit will be useful inconducting the diagnostic assays discussed herein, e.g., in determiningthe status of treatment of an illness characterized by inappropriatecell target or soluble analyte expression in a blood sample. Such adiagnostic kit contains the dyes, ligands, capture medium, and othercomponents of the methods of this invention. Such kits also containlabels, exemplified above, pre-attached to the other components of thespecific assay to be performed, or provided separately for attachment toa selected component, e.g., a substrate. Alternatively, such kits maycontain a simple mixture of such compositions or means for preparing asimple mixture.

Such kits provide a convenient, efficient way for a clinical laboratoryto screen blood samples or other biological samples containing cellsaccording to this invention.

One of skill in the art may be expected to vary the components of thesediagnostic kits in obvious ways based on the knowledge in the artcoupled with this disclosure. Such varied components are included inthis embodiment of the invention.

The kit further comprising at least one of the following additionalcomponents selected from the group consisting of suitable vessels forcontaining samples, suitable controls or tables of normal ordisease-characteristic values of activated platelets; an anti-coagulantor coagulation pathway inhibitor, other reagents suitable for theperformance of flow cytometric analyses and combinations thereof;suitable diluents and buffers for the samples, disposable gloves,decontamination instructions, applicator sticks or containers, andsample preparatory cups.

EXAMPLES

These examples demonstrate the use of the methods and compositions ofthe invention and the analysis thereof. The data reported in theseExamples demonstrates that the novel methods of this invention haveperformance parameters that permit improved efficient and substantiallysimultaneous analysis of samples with multiple types of targets. Theseexamples are illustrative and do not limit the scope thereon. One ofskill in the art will appreciate that although specific reagents andconditions are outlined in the following examples, modifications asdescribed above can be made to provide the compositions of thisinvention or processes for use thereof.

Example 1

ImmunoPlex Multiple Analyte Sandwich Immunoassay.

The following example was performed to test combined use of flowcytometry and immunoassay technology for substantially simultaneousassay of cellular marker expression profile (immunophenotyping),quantitation of soluble (serum markers), and white blood cell percentagein a single assay tube.

To a sample (100 uL) of EDTA-treated whole blood are added the followingreagents:

-   -   (a) The capture medium is 50 μl of a six-bead polystyrene        microsphere bead population with distinct fluorescence        intensities. The microspheres or beads are generally larger than        3.6 μm and smaller than 10 μm. Antibodies are individually        covalently attached to a subset of the beads by conventional        methods to create the antibody-conjugated fluorescence capture        beads with bead specificity for IL-2, IL-4, IL-5, IL-10, TNF-α        or IFNγ.    -   (b) Phycoerythrin (PE)-conjugated anti-human detector soluble        ligand (50 ul/test) with an antibody specificity for anti-IL-2,        anti-IL-4, anti-IL-5, anti-IL-10, anti-TNF-α, or anti-IFN-γ.    -   (c) A soluble ligand to the cellular target, i.e., 20 μl of        anti-CD45-FITC (Beckman Coulter).

This reagent mixture is incubated for between about 1 hour to 3 hours atroom temperature with gentle mixing and also is protected from light.For comparison, a parallel sample was created by substituting plasma forwhole blood.

Once incubated, the red blood cells in the sample were lysed by theaddition of ImmunoPrep™ reagents (Beckman Coulter). The samples werethen subjected to cytometric flow analysis using a Beckman Coulter FC500 Flow Cytometer without any further manipulation or separation of thevarious complexes formed among the reagents in the sample and the datawas collected.

The results showed no significant difference in cytokine values whencapture bead/detector reagents were incubated in plasma or in wholeblood, with or without fluorochrome anti-CD markers. The assay range forthe cytokines evaluated was from 0 to 5000 pg/mL. There was also noeffect of the beads on cellular scatter parameters or on thedetermination of CD expression, as measured by mean fluorescenceintensity. Thus the data provided reliable information on white bloodcell percentages, cell surface expression of marker proteins, and serumcytokine levels in a single tube analysis format.

Example 2

ImmunoPlex Single Analyte Capture Immunoassay

A sample of whole blood (100 μl) collected in the anticoagulant EDTA, iskept on ice or at room temperature throughout this experiment. Parallelsamples were created by substituting plasma or buffer for the wholeblood. To these samples were added the following reagents:

-   -   (a) 10 μl of capture beads. As capture bead, a single        non-fluorescent, paramagnetic, polystyrene microsphere is used        to create six populations with bound antibodies to the soluble        analyte IL2, i.e., human IL-2 antibodies. The beads are larger        than 3.61 μm and smaller than 10 μm.    -   (b) 20 μl of a ligand to the cellular target CD14, which is an        antibody labeled with fluorescent isothiocyanate        (anti-CD14-FITC) (Beckman Coulter); and    -   (c) 10 μl of a ligand to the cellular target CD45, which is an        antibody labeled with phycocyanin-5 (anti-CD45-PC5) (Beckman        Coulter).

The samples are then incubated for 60 minutes mixing twice every 30seconds (or alternatively rocking).

Thereafter 10 μl of a soluble ligand to the soluble analyte IL-2, i.e.,phycoerythrin (PE) conjugated anti-IL2 reporter antibody, was added tothe samples of buffer, plasma and whole blood and the samples wereincubated for 30 minutes, again mixing twice per 30 seconds (orrocking).

The whole blood samples were then lysed using ImmunoPrep™ reagents(Beckman Coulter) and all samples were subjected to cytometric flowanalysis on a Cytomics FC500 or Coulter® XL/MCL™ flow cytometer withoutany further manipulation or separation of the various complexes formedamong the reagents in the sample and the data was collected.

To assess bead effect on light scatter and CD expression, the sampleswere processed with and without beads to assess any impact on cellularlight scatter patterns and mean fluorescent intensity (MFI).Lymphocytes, monocytes and granulocytes were examined, as beingconsidered worst case scenario due to potential phagocytosis ofparticles.

The data collected showed there was no difference in values with respectto light scatter and MFI obtained in plasma compared to whole blood,confirming that phagocytosis of beads was inhibited.

The assay was performed with and without anti-IL-2 beads, with andwithout soluble target antibody (IL2-PE), and with and without cellulartarget antibody (CD14-FITC). Isotypic controls were used as negativecontrols. As expected, a distinctly different regression equation wasseen from plotting the results obtained when the sample media was bufferas compared to plasma or whole blood, indicating “matrix effects”. Theaddition of beads and soluble target antibody did not adversely impactcellular scatter pattern or antigenic expression.

Example 3

ImmunoPlex CD20

This example and FIG. 4 illustrate use of the invention methods tomonitor treatment of a patient with a ligand (antibody) that binds toCD20. Whole blood, fine needle aspirates or bone marrow is placed in ananalysis tube and components are stained by coincubation with thefollowing components:

-   -   a) an antibody that binds specifically to the cell surface        expressed target, CD20, and which has been conjugated to a first        distinguishable fluorescent label, fluorochrome 1 (Ab-FL1)        (clone HRC20)-PE);    -   b) a second antibody that identifies the cell lineage (i.e.        CD19) and which is conjugated to a second distinguishable        fluorescent label, fluorochrome 2 (Ab-FL2)(CD19-ECD®) a direct        antibody conjugate; and    -   c) a capture bead (as described in Example 2 above) that can be        discriminated from cells by physical or fluorescent        characteristics and is linked to CD20 antigen, for example,        covalently (and/or a preserved cell that has a known expression        of CD20 and which can be discriminated from all other cells in        the mixture by physical and/or fluorescent characteristics). The        mixture is incubated for a sufficient time to allow optimal        binding of components in the analysis tube. Excess (unbound)        components a) and b) and non-bound plasma components are removed        through washing.

The sample is then treated with a fixation and permeabilization reagent(e.g. IntraPrep™ reagent, Beckman Coulter) to allow entry into cells fortagging of intracellular marker proteins, and an antibody that bindsspecifically to intracellular CD20 and which has been conjugated to athird distinguishable fluorescent label, fluorochrome 3 (Ab-FL3) (cloneL26)-FITC) is introduced into the analysis tube (Mason, D. Y. et al.1990. Am J Pathol 136:1215-1222). Incubation proceeds to allow optimalbinding of Ab-FL3. The sample may be lysed and/or washed and is thenanalyzed on a flow cytometer or imaging system to enable the comparisonand relative quantitation of cell surface expression, intracellularexpression, as well as circulating CD20 expression by comparison of therelative fluorescent staining of a combination of one or more beadand/or preserved cell combinations using the procedure illustratedschematically in FIG. 4. Utilization of calibrator beads or cells canfurther enable a quantitative measurement to be made. Additionallyhematology differential parameters can be ascertained.

Example 4

This example and FIG. 5 illustrate the invention assay procedure as usedto monitor the course of treatment of a patient using CD52 antibody,CAMPATH-1. Whole blood, bone marrow or single cell suspensions are firstplaced in a single container and incubated with a first captureparticle, such as a distinguishable capture bead, that has beencovalently linked to a synthetic CD52 antigen (1Bead-Ag) that containsthe epitope to which CAMPATH-1 binds, and with a second capture particlethat is covalently linked to CAMPATH-1, the treatment antibody(2Bead-Drug). The order of introduction of the two is immaterial. Themixture is incubated (first incubation period) for a sufficient time toallow optimal binding of the capture beads with their targets in thesample. Bead-Ag will bind with any circulating drug, Bead-Drug will bindwith any circulating anti-CAMPATH-1 antibodies or shed receptor in thesample.

For analysis, the sample is then stained in the analysis tube with aseries of antibodies labeled with distinguishable fluorochromes:

-   -   a) A treatment antibody that binds specifically to the cell        surface expressed target, e.g. CD52 conjugated to first        fluorescent label FL2 (CAMPATH-1-PE)(Ab-FL2);        b) a second antibody that binds the same expressed target        antigen (CD52) at a different site than CAMPATH-1 and which is        conjugated to a second fluorescent label (HI186)—PC7) (Ab-FL5);    -   c) To detect any target soluble analyte, a third antibody that        binds specifically to human immunoglobulin (e.g.        anti-HuIgG-FITC), and which is conjugated to a third fluorescent        label (Ab-FL1); and    -   d) A fourth marker for cell lineage may also be added at this        time (Ab-FL4; e.g. CD19-ECD (not shown in FIG. 5).

The sample is incubated in the analysis tube (i.e. second incubationperiod) for a sufficient time to allow optimal binding betweencomponents. The sample is then lysed and/or washed to remove excessAb-FLs, non-bound plasma components and red cells and the contents ofthe container are analyzed on a flow cytometer. The amounts of CD52present on the cell surface and circulating in the subject's blood canbe assessed along with the appearance of any anti-CAMPATH-1 response.Utilization of calibrator beads or cells further enables a quantitativemeasurement to be made. The entire analysis is performed on the sameinstrument and without separating the complexes formed in the sampletube. Additionally hematology differential parameters can beascertained.

Example 5

This example illustrates use of the invention methods to monitortreatment of a patient with heparin. Whole blood is first stabilized(ThombFix, or CTAD (citrate, theophylline, adenosine, and dipyridamolecocktail) (Becton Dickenson, CA) to prevent any artifactual activation.A bead is covalently linked to heparin:platelet factor 4 (H:PF4)complexes and/or to a preserved cell with H:PF4 and which can bediscriminated from all other cells in the mixture by physical and/orfluorescent characteristics (Bead-H:PF4). A fluorochrome-conjugatedantibody (Ab-FL1) that binds specifically to a platelet activationantigen (e.g. CD62p-FITC) is prepared. The beads andfluorochrome-conjugated antibody are added to the stabilized blood in ananalysis tube. A second fluorochrome-conjugated antibody that bindsspecifically to an additional platelet marker (e.g. CD41-PC7) may alsobe added to the analysis tube at this time (Ab-FL4;). The sample isincubated for a sufficient time to allow optimal binding of Bead-H:PF4with any anti-H:PF4 autoantibodies. A subsequent wash removes any excessAb-FL1, Ab-FL2 or non-bound plasma components. To detect the anti-H:PF4autoantibodies bound by the Bead-H:PF4, a third antibody that bindsspecifically to human immunoglobulins conjugated to a third fluorescentlabel FL2 (anti-HuIg-PE) is added to the analysis tube and the mixtureis incubated to allow binding to the full extent to occur.

The sample is then analyzed on a flow cytometer using the procedureillustrated schematically in FIG. 6. Detection of platelet activation isassessed by determining the percent of CD62p, autoantibody measurementis quantified by the mean fluorescent intensity of the complex includingBead-H:PF4:anti-H:PF4:anti-HuIg-PE (FL2). Platelet concentration isdetermined by the ratio of the red cells to the platelets as per theguidelines of the International Society for Laboratory Hematology (ISLH)(International Council for Standardization in Haematology Expert Panelon Cytometry and International Society of Laboratory Hematology TaskForce on Platelet Counting. Platelet counting by the RBC/platelet ratiomethod: a reference method. Am J Clin Pathol. 2001; 115:460-464) Withoutsample separation prior to analysis, platelet count, platelet activationand presence of anti-H:PF4 antibodies are analyzed in a single tube forthe determination of HIT. (PLT=platelets; RBC=red blood cells).

All documents cited above are incorporated by reference herein. Thecompositions and processes of the present invention are encompassed bythe scope of the claims appended hereto.

REFERENCE LIST

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1-62. (canceled)
 63. A method for evaluation of a biological samplecontaining at least one cell type bearing at least one cellular targetand at least one soluble analyte, the steps comprising: (a) adding to asingle container said sample with (i) at least one soluble ligand thatbinds to said cellular target, (ii) at least one soluble ligand thatbinds to said soluble analyte, or at last one competing soluble analyte;and (iii) a solid phase capture medium that binds directly to saidsoluble analyte, indirectly to said soluble analyte, or to said solubleligand that binds to said soluble analyte; and (b) simultaneouslyanalyzing said sample (a) without physically separating the complexescomprising a complex that forms between said cellular target and said atleast one soluble ligand, and a complex that forms between said capturemedium bound directly to said soluble analyte, or between said capturemedium bound indirectly to said soluble analyte, or between said capturemedium bound to said soluble ligand that is bound to said solubleanalyte.
 64. The method of claim 63, further comprising in (a): (i)adding to said sample a solid phase capture medium on which areimmobilized multiple first ligands that are capable of binding to saidsoluble analyte, and wherein said capture medium, said first ligand, andsaid soluble analyte form a first complex; (ii) adding to sampleobtained from (i) a soluble second ligand that binds specifically tosaid cellular target, wherein each second ligand is associated with afirst detectable label, and wherein multiple second ligands can bind toa single target cell to form a second complex; (iii) adding to sampleobtained from (ii) a third ligand associated with a second detectablelabel and that binds specifically to said first complex, wherein saidthird ligand and said first complex form a third complex, and (b)simultaneously analyzing sample obtained from (aiii) without physicallyseparating said complexes by discriminating between said third complexand said second complex.
 65. The method of claim 63, wherein saidsoluble analyte is selected from the group consisting of a serum marker,a protein, a virus, a hormone, a lipid, a nucleic acid sequence, acarbohydrate, a pharmaceutical drug, a toxin, and an antigen shed from acell.
 66. The method of claim 63, wherein said method is useful indiagnosis of a disease or condition selected from the group consistingof sepsis, inflammation, autoimmune disease, cardiovascular disease,viral infection, bacterial infection, and drug interaction.
 67. A methodfor diagnosing sepsis or monitoring the progress thereof by performingthe method of claim 63, wherein said cellular target is selected fromthe group consisting of CD64 (N), HLA-DR (Mo), CD11a, CD14/CD16, CD142;and said soluble target is selected from the group consisting of IL-6,IL-10, IL-1, TNF-alpha, neopterin, C-reactive protein, procalcitonin,and activated Protein C.
 68. A method for diagnosing autoimmune diseaseor monitoring the progress thereof by performing the method of claim 63,wherein said cellular target is selected from the group consisting ofactivated T cells and activated B cells; and said soluble target isselected from the group consisting of C-reactive protein, a chemokine,and a cytokine.
 69. A method for diagnosing cardiovascular disease ormonitoring the progress thereof by performing the method of claim 63,wherein said cellular target is selected from the group consisting ofplatelet-leucocyte aggregates, CD142 (TF); and said soluble target isselected from the group consisting of hsC-reactive protein, troponin,and myoglobin.
 70. A method for differential diagnosis of viral andbacterial infections or monitoring the progress thereof by performingthe method of claim 63, wherein said cellular target is selected fromthe group consisting of HLA-DR, CD4/CD8, CD64 (N), CD14/CD16; and saidsoluble target is selected from the group consisting of IFN-gamma,neopterin, and C-reactive protein.
 71. A method for monitoring treatmentof a patient in need thereof with a treatment ligand that bindsspecifically to the cell surface expressed target CD20, comprising: a)obtaining a container containing a sample comprising bodily fluidcontaining CD20⁺ cells obtained from the patient; b) performing one ofthe following: i) incubating assay components in the container with afirst soluble ligand that binds specifically to CD20 and is conjugatedto a first distinguishable fluorescent label under conditions and for atime to allow formation of complexes of CD20 and the first ligand; orii) adding to the container a second soluble ligand that bindsspecifically to B cells and is conjugated to a second fluorescent labelunder conditions and for a time to allow formation of complexes betweenassay components; or iii) the following combination of steps: adding tothe container a capture particle linked to CD20 antigen; permeabilizingcells in the container; and incubating assay components in the containerwith a third soluble ligand that binds specifically to intracellularCD20 and is conjugated to a third distinguishable fluorescent labelunder conditions and for a time to allow formation of complexes ofintracellular CD20 and the first third ligand; and c) detecting thepresence of fluorescence from fluorescent labels in complexes formed inthe container to monitor the treatment of the patient.
 72. The method ofclaim 71, wherein performance of b) iii) is selected, furthercomprising, prior to performance of b) iii), adding to the container: a)the second soluble ligand that binds specifically to B-cells conjugatedto the second distinguishable fluorescent label; and/or a third solubleligand that binds specifically to CD20⁺ cells, and/or the captureparticle that contains the CD20 antigen; and b) incubating contents ofthe container under conditions and for a time sufficient to allowcomplex formation between assay components therein.
 73. The method ofclaim 71, wherein the sample is whole blood.
 74. The method of claim 71,wherein the ligands are antibodies.
 75. The method of claim 71, whereinthe treatment ligand is Rituximab® or Bexxar™ monoclonal antibody. 76.The method of claim 71, wherein the relative intensities of thefluorescent labels in the complexes are detected by a fluorometer orflow cytometer.
 77. A method for monitoring treatment of a patient inneed thereof with a treatment ligand that binds specifically to the cellsurface expressed target CD52, comprising: a) obtaining a containercontaining a sample comprising a bodily fluid containing CD52⁺ cellsobtained from the patient; b) incubating the sample in the containerunder conditions and for a time sufficient to allow complex formationwith: i) a first ligand that binds specifically to the expressed targetat the binding site of the treatment ligand conjugated to a firstdistinguishable fluorescent label and ii) one assay component selectedfrom: a) a second ligand that binds the expressed target at a differentbinding site than the treatment ligand conjugated to a seconddistinguishable ligand; b) a third ligand that binds specifically tohuman immunoglobulin conjugated to a third distinguishable fluorescentlabel; and c) a first distinguishable capture particle linked to a CD52antigen; and d) a second distinguishable capture particle linked to thetreatment ligand; and c) detecting substantially simultaneouslyfluorescence from the fluorescent labels in the complexes formed in thecontainer to monitor the treatment of the patient.
 78. The method ofclaim 77, wherein in b)ii) the second ligand is selected, said methodfurther comprising: in a) adding to the container either the firstcapture particle or a second distinguishable capture particle linked tothe treatment ligand; or in b ii) adding the third ligand and either oneor both of the first capture particle and the second capture particle.79. The method of claim 77, wherein in b)ii) the second ligand isselected, the method further comprising in a), adding to the containerone or both of the first capture particle and a second distinguishablecapture particle linked to the treatment ligand.
 80. The method of claim77, wherein in b)ii) the third ligand is selected, the method furthercomprising in a), adding to the container one or both of a firstdistinguishable capture particle linked to a CD52 antigen and the secondcapture particle.
 81. The method of claim 77, wherein the treatmentligand is CAMPATH-1H monoclonal antibody and the first ligand is anantibody that binds specifically to an epitope of CD52 that is differentfrom the epitope bound by CAMPATH-1.
 82. The method of claim 77, whereinthe treatment is treatment of B-cell chronic lymphocytic leukemia.
 83. Amethod for monitoring side effects of heparin therapy in a patient inneed thereof, said method comprising: a) incubating the following assaycomponents in a container under conditions and for a time sufficient toallow complex formation between: i) a sample comprising whole blood ofthe patient; ii) a distinguishable capture particle linked toheparin:platelet factor 4 complex; iii) a first soluble ligand thatbinds specifically to a platelet activation antigen and is conjugated toa first fluorescent label; and iv) a second soluble ligand that bindsspecifically to platelets and is conjugated to a second fluorescentlabel; b) incubating in the container under conditions and for a timesufficient to allow complex formation between the complexes formed in a)and a third ligand that binds specifically to human immunoglobulins andis conjugated to a third distinguishable fluorescent label, therebyforming a mixture of complexes therein; and c) detecting fluorescencefrom the first fluorescent label, second fluorescent label or the thirdfluorescent label in the mixture of complexes to monitor heparin therapyin the patient.
 84. The method of claim 83, wherein the sample compriseswhole blood or platelet-rich plasma.
 85. The method of claim 83, whereinthe ligands are antibodies.
 86. The method of claim 83, wherein thedetecting is by fluorometer or flow cytometer.
 87. A kit comprising: a)at least one soluble ligand that binds a cellular target in a liquidsample; b) at least one soluble ligand that binds a soluble analyte insaid sample or at least one soluble analyte associated with a detectablelabel; and c) a solid phase capture medium that binds directly to saidsoluble analyte, indirectly to said soluble analyte, or to said solubleligand that binds to said soluble analyte.
 88. The kit of claim 87,further comprising a container comprising one or a mixture of componentsa) through c).
 89. The kit of claim 87, wherein the kit is formonitoring treatment of a patient with a treatment ligand that bindsspecifically to the cell surface expressed target CD20, and comprises:a) a first soluble ligand that binds specifically to CD20; and one ormore of the following: b) a second soluble ligand conjugated that bindsspecifically to intracellular CD20+ cells; c) a third soluble ligandthat binds specifically to B cells; and d) a capture particle linked toCD20 antigen.
 90. The kit of claim 87, wherein the kit is for monitoringtreatment of a patient with a treatment ligand that binds specificallyto CD52 antigen and comprises a) a first distinguishable captureparticle linked to a CD52 antigen; and b) a first soluble ligand thatbinds specifically to the expressed target at the binding site of thetreatment ligand conjugated to a first distinguishable fluorescentlabel.
 91. The kit of claim 87, wherein the kit is for monitoringheparin therapy of a patient and comprises: a) a distinguishable captureparticle linked to a heparin:platelet factor 4 complex; and one or moreof the following: b) a first soluble ligand that binds specifically to aplatelet activation antigen; c) a second soluble ligand that bindsspecifically to platelets; and d) a third soluble ligand that bindsspecifically to human immunoglobulins.
 92. The method of claim 63,wherein said capture medium is a physiologically compatible bead.
 93. Amethod for monitoring treatment of a patient in need thereof with atreatment ligand that binds specifically to the cell surface expressedantigen, comprising: a) obtaining a container containing a samplecomprising bodily fluid containing the antigen obtained from thepatient; b) performing one of the following: i) incubating assaycomponents in the container with a first soluble ligand that bindsspecifically to a cell surface antigen and is conjugated to a firstdistinguishable fluorescent label under conditions and for a time toallow formation of complexes of target and the first ligand; or ii)adding to the container a second soluble ligand that binds specificallyto a defined cell lineage specific antigen and is conjugated to a secondfluorescent label under conditions and for a time to allow formation ofcomplexes between assay components; or iii) the following combination ofsteps: adding to the container a capture particle linked to thetarget-antigen; and incubating assay components in the container with athird soluble ligand distinct from the first soluble ligand and thatbinds specifically to a separate, distinct epitope on the cell surfaceantigen and is conjugated to a third distinguishable fluorescent labelunder conditions and for a time to allow formation of complexes of cellsurface antigen and the third ligand; and c) detecting the presence offluorescence from fluorescent labels in complexes formed in thecontainer to monitor the treatment of the patient.
 94. A method formonitoring treatment of a patient in need thereof with a treatmentligand that binds specifically to intracellular expressed targetantigen, comprising: a) obtaining a container containing a samplecomprising bodily fluid containing antigen obtained from the patient; b)performing one of the following: i) adding to the container a firstsoluble ligand that binds specifically to a defined cell lineagespecific antigen and is conjugated to a first fluorescent label underconditions and for a time to allow formation of complexes between assaycomponents; or ii) the following combination of steps: adding to thecontainer a capture particle linked to antigen; permeabilizing cells inthe container; and iii) incubating assay components in the containerwith a second soluble ligand that binds specifically to theintracellular antigen of interest and is conjugated to a seconddistinguishable fluorescent label under conditions and for a time toallow formation of complexes of and the second ligand; or iv) incubatingassay components in the container with a third soluble ligand distinctfrom the first soluble ligand that also binds specifically to aseparate, distinct epitope on the intracellular antigen of interest andis conjugated to a third distinguishable fluorescent label underconditions and for a time to allow formation of complexes of theintracellular antigen and the third ligand; and c) detecting thepresence of fluorescence from fluorescent labels in complexes formed inthe container to monitor the treatment of the patient.
 95. The method asin claim 93 or 94, wherein the antigen is CD20 or CD52.